Combination therapy for osteoporosis

ABSTRACT

Pharmaceutical combination compositions including certain estrogen agonists/antagonists and prostaglandins or prostaglandin agonists/antagonists. The compositions are useful for the treatment of bone disorders including osteoporosis.

BACKGROUND OF THE INVENTION

[0001] This invention relates to a pharmaceutical combination ofestrogen agonists/antagonists and agents that stimulate bone formationand increase bone mass, kits containing such combinations and the use ofsuch combinations to treat conditions which present with low bone massin mammals, including humans.

[0002] Osteoporosis is a systemic skeletal disease, characterized by lowbone mass and deterioration of bone tissue, with a consequent increasein bone fragility and susceptibility to fracture. In the U.S., thecondition affects more than 25 million people and causes more than 1.3million fractures each year, including 500,000 spine, 250,000 hip and240,000 wrist fractures annually. Hip fractures are the most serious,with 5-20% of patients dying within one year, and over 50% of survivorsbeing incapacitated.

[0003] The elderly are at greatest risk of osteoporosis, and the problemis therefore predicted to increase significantly with the aging of thepopulation. Worldwide fracture incidence is forecast to increasethreefold over the next 60 years, and one study estimates that therewill be 4.5 million hip fractures worldwide in 2050.

[0004] Women are at greater risk of osteoporosis than men. Womenexperience a sharp acceleration of bone loss immediately followingmenopause. Other factors that increase bone loss leading to osteoporosisinclude smoking, alcohol abuse, a sedentary lifestyle and low calciumintake.

[0005] Estrogen is the agent of choice in preventing osteoporosis orpost menopausal bone loss in women. In addition, Black, et al. in EP06051931A1 report that estrogen, particularly when taken orally, lowersplasma levels of LDL and raises those of the beneficial high densitylipoproteins (HDL's). Long-term estrogen therapy, however, has beenimplicated in a variety of disorders, including an increase in the riskof uterine cancer, endometrial cancer and possibly breast cancer,causing many women to either avoid this treatment or take the medicationfor only a short period of time. Although the risk of endometrial canceris thought to be reduced by a concurrent use of a progesterone, there isstill concern about possible increased risk of breast cancer with theuse of estrogen. Recently suggested therapeutic regimens, which seek tolessen the cancer risk, such as administering combinations ofprogesterone and estrogen, cause the patient to experience unacceptablebleeding. Furthermore, combining progesterone with estrogen seems toblunt the serum cholesterol lowering effects of estrogen. Thesignificant undesirable side effects associated with estrogen therapysupport the need to develop alternative therapies for osteoporosis thathave the desirable beneficial effect on serum LDL but do not causeundesirable side effects.

[0006] Recently, a number of estrogen agonists/antagonists have beenproposed for treatment of osteoporosis. It has been reported(Osteoporosis Conference Scrip No. 1812/13 Apr. 16/20, 1993, p. 29) thatraloxifene, 6hydroxy-2-(4-hydroxyphenyl)-3-[4-(2-piperidinoethoxy)benzoyl]benzo[b]thiophene, mimics the favorableaction of estrogen on bone and lipids but, unlike estrogen, has minimaluterine stimulatory effect. [Black, L. J. et al., Raloxifene (LY139481Hcl) Prevents Bone Loss and Reduces Serum Cholesterol Without CausingUterine Hypertrophy in Ovariectomized Rats, J. Clin. Invest., 1994,93:6369].

[0007] Also, tamoxifen,1-(4-β-dimethylaminoethoxyphenyl)-1,2-diphenyl-but-1-ene, is anantiestrogen that is proposed as an osteoporosis agent which has apalliative effect on breast cancer, but is reported to have someestrogenic activity in the uterus. Gill-Sharma, et al., J. Reproductionand Fertility (1993) 99, 395, disclose that tamoxifen at 200 and 400mg/kg/day reduces the weights of the testes and secondary sex organs inmale rats.

[0008] In addition U.S. Pat. No. 5,254,594 (the disclosure of which ishereby incorporated by reference) discloses the use of droloxifene forthe treatment of bone diseases including osteoporosis.

[0009] Agents such as droloxifene prevent bone loss and thereby reducethe risk of fracture without estrogen's side effects. However, estrogenand estrogen agonists alone are only expected to reduce the fracturerisk by about 50% leaving approximately 50% of ostepenic women still atrisk for an osteoporotic fracture.

[0010] Nonestogen agonists/antagonists such as bisphosphonates are alsoproposed for the treatment of osteoporosis. For example, Fosanax® is abisphosphonate that is currently marketed for the treatment ofosteoporosis. Other bisphosphonates currently undergoing regulatoryreview include risedronate, tiludronate, and ibandronate.

[0011] Frost et al. in “Treatment of Osteoporosis by Manipulation ofCoherent Bone Cell Populations”, Clinical Orthopedics and RelatedResearch, 143, 227 (1979) discloses a theoretical model that suggests itshould be possible to synchronize the activity and metabolism of bonecells by administering a bone cell activating agent first, followed by abons resorption inhibiting agent and then normal bone formation isallowed to occur.

[0012] Tang et al., Restoring and Maintaining Bone in Osteogenic FemaleRat Skeleton: I. Changes in Bone Mass and Structure, J. Bone MineralResearch 7 (9), p1093-1104, 1992 discloses data for the lose, restoreand maintain (LRM) concept, a practical approach for reversing existingosteoporosis. The LRM concept uses anabolic agents to restore bone massand architecture (+phase) and then switches to an agent with theestablished ability to maintain bone mass, to keep the new bone(+/−phase). The rat study utilized PGE₂ and risedronate, abisphosphonate, to show that most of the new cancellous and corticalbone induced by PGE₂ can be maintained for at least 60 days afterdiscontinuing PGE₂ by administering risedronate.

[0013] Combinations of bisphosphonates and prostaglandins for thetreatment of osteoporosis are disclosed. E.P. App. No. 0 381 296 teachesthe use of a kit wherein a bone activating period or treatment regime isfollowed by a bone resorption inhibiting regime. Examples of boneactivating compounds cited in this reference include parathyroid hormone(PTH), inorganic phosphate, growth hormone, fluoride, thyroid hormone(e.g., thyroxin), certain vitamin D metabolites and prostaglandins (PGE₂in a dose regime of 10 mg/kg per day). Polyphosphonates are disclosed asthe bone resorption inhibiting agents.

[0014] PCT/US93/08529 discloses the simultaneous delivery of a boneactivating agent such as a prostaglandin that is chemically coupled to abone resorption inhibiting compound which selectively delivers the boneactivating agent to the target area. Upon gradual hydrolysis of thenovel compound, the hydrolyzed products are able to provide boneresorption inhibiting activity (via the bisphosphonates) and bone growthor stimulating activity (via PGE₂).

[0015] The effects of a combination of prostaglandin E2 and risedronate(a bisphosphonate) was studied in Lin et al., Effects of ProstaglandinE2 and Risedronate Administration on Cancellous Bone in Older FemaleRats, Bone 15 (5), p489-496, 1994.

[0016] Qiu et al., Experimental Study on Antiatherosclerotic Treatmentby PGE ₂ Combined With Vitamin E and Estradiol, Chinese Medical Journal,108 (1) p33-36, 1995 disclose that a single dose of PGE₂ combined withvitamin E and with estradiol had more coordinative inhibition on aorticand coronary atherosclerotic lesions, as well as on plateletaggregation, smooth muscle cell proliferation and lipid peroxidationthan that of a single dose of PGE₂.

[0017] The abstract for “Nonhormnonal Alternatives for the Management ofEarly Menopause in Younger Women with Breast Cancer”, Monogr. Nat.Cancer Inst. (16), 161-167, 1994, states “The use of several nonestrogenapproaches for the prevention and treatment of osteoporosis has beenpromising. Traditional recommendations to maintain skeletal integrity,such as weight-bearing exercise; a diet rich in calcium and limited incaffeine, alcohol, and protein; avoidance of smoking; and measures tominimize trauma have been expanded to include the use or investigationof drugs (either alone or in combination). These drugs includeprogestins, vitamin D metabolites, injectable and intranasal syntheticsalmon calcitonin, bisphosphonates, sodium fluoride, parathyroidhormone, growth factors, tamoxifen, etc.”

[0018] Thus, although there exist a variety of osteoporosis therapiesthere is a continuing need and a continuing search in this field of artfor alternative therapies due to only limited success of currenttherapies in reducing osteoporotie fractures.

SUMMARY OF THE INVENTION

[0019] This invention is directed to a pharmaceutical compositionincluding estrogen agonists/antagonists and anabolic agents and for theuse of such compositions for the treatment of conditions which presentwith low bone mass, including osteoporosis in mammals (e.g., humans,particularly women).

[0020] The combination comprises a therapeutically effective amount of afirst compound, said first compound being an estrogen agonlisantagonist;and a therapeutically effective amount of a second compound, said secondcompound being a prostaglandin or a prostaglandin agonist/antagonist.

[0021] Preferred estrogen agonist/antagonists include droloxifene,raloxifene, tamoxifen, 4-hydroxy-tamoxifen,

[0022]Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0023](−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0024]Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0025]Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;

[0026]1-(4′-Pyrrolidinoethoxyphenyl)-2-(4′-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0027]Cis-6-(4-hydroxyphenyl)-5-[4-(2-pipeidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;and

[0028]1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline.

[0029] Preferred anabolic agents include PGD₁, PGD₂, PGE₂, PGE₁, PGF₂,PGF₂α and3S-(3-Hydroxyphenyl-4-phenyl-butyl)-2R-[6-(1H-tetrazol-5-yl)-hexyl]-cyclopentanone.

[0030] Another aspect of this invention is a method for treating mammalswhich present with low bone mass comprising administering to a mammalhaving a condition which presents with low bone mass

[0031] a. a therapeutically effective amount of a first compound, saidfirst compound being an estrogen agonist/antagonist; and

[0032] b. a therapeutically effective amount of a second compound, saidsecond compound being a prostaglandin or a prostaglandinagonist/antagonist.

[0033] Preferred estrogen agonist/antagonists in this method includedroloxifene, raloxifene, tamoxifen, 4-hydroxy-tamoxifen,

[0034]Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0035](−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0036]Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0037]Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;

[0038]1-(4′-Pyrroldinoethoxyphenyl)-2-(4′-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0039]Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene2-ol;and

[0040]1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline.

[0041] Preferred anabolic agents in this method include PGD₁, PGD₂,PGE₂, PGE₁, PGF₂, PGF₂α and3S-(3-Hydroxy-4-phenyl-butyl)-2R-[6-(1H-tetrazol-5-yl)-hexyl]-cyclopentanone.

[0042] A preferred aspect of this method is wherein the condition whichpresents with low bone mass is osteoporosis.

[0043] Another preferred aspect of this method is wherein the firstcompound and the second compound are administered substantiallysimultaneously.

[0044] Another preferred aspect of this method is wherein the secondcompound is administered for a period of from about three months toabout three years.

[0045] Optionally the administration of the second compound is followedby administration of the first compound for a period of from about threemonths to about three years without the administration of the secondcompound during the second period of from about three months to aboutthree years.

[0046] Alternatively, the administration of the second compound isfollowed by administration of the first compound for a period greaterthan about three years without the administration of the second compoundduring the greater than about three year period.

[0047] Another aspect of this invention is a synergistic pharmaceuticalcomposition comprising

[0048] a. an amount of a first compound, said first compound being anestrogen agonist/antagonist; and

[0049] b. an amount of a second compound, said second compound being aprostaglandin or a prostaglandin agonist/antagonist

[0050] wherein the amount of the first compound alone and the amount ofthe second compound alone is insufficient to achieve the therapeuticeffects of increase in bone formation and decrease in bone resorption ifadministered simultaneously and wherein the combined effect of theamounts of the first and second compounds is greater than the sum of thetherapeutic effects achievable with the individual amounts of the firstand second compound, and a pharmaceutically acceptable diluent orcarrier.

[0051] Yet another aspect of this invention is a synergistic method fortreating mammals which present with low bone mass comprisingadministering to a mammal having a condition which presents with lowbone mass

[0052] a. an amount of a first compound, said first compound being anestrogen agonist/antagonist; and

[0053] b. an amount of a second compound, said second compound being aprostaglandin or a prostaglandin agonist/antagonist

[0054] wherein the amount of the first compound alone and the amount ofthe second compound alone is insufficient to achieve the therapeuticeffects of increase in bone formation and decrease in bone resorption ifadministered simultaneously and wherein the combined effect of theamounts of the first and second compounds is greater than the sum of thetherapeutic effects achievable with the individual amounts of the firstand second compound, and a pharmaceutically acceptable diluent orcarrier.

[0055] Another aspect of this invention is a kit containing a treatmentfor a condition which presents with low bone mass comprising:

[0056] a. a therapeutically effective amount of an estrogenagonist/antagonist and a pharmaceutically acceptable carrier in a firstunit dosage form;

[0057] b. a therapeutically effective amount of a prostaglandin or aprostaglandin agonist/antagonist and a pharmaceutically acceptablecarrier in a second unit dosage form; and

[0058] c. container means for containing said first and second dosageforms.

[0059] Another aspect of this invention is directed to a pharmaceuticalcomposition comprising:

[0060] a. a therapeutically effective amount of a first compound, saidfirst compound being droloxifene, raloxifene, tamoxifen or idoxifene;and

[0061] b. a therapeutically effective amount of a second compound, saidsecond compound being sodium fluoride orN-[1-(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677.

[0062] A preferred aspect of this composition is wherein the firstcompound is droloxifene.

[0063] Another aspect of this invention is directed to a method fortreating mammals which present with low bone mass comprisingadministering to a mammal having a condition which presents with lowbone mass

[0064] a. a therapeutically effective amount of a first compound, saidfirst compound being droloxifene, raloxifene, tamoxifen or idoxifene;and

[0065] b. a therapeutically effective amount of a second compound, saidsecond compound being sodium fluoride orN-[1-(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677.

[0066] A preferred aspect of this method is wherein the first compoundis droloxifene.

[0067] Another preferred aspect of this method is wherein the conditionwhich presents with low bone mass is osteoporosis.

[0068] Another preferred aspect of this method is wherein the firstcompound and the second compound are administered substantiallysimultaneously.

[0069] Another preferred aspect of this method is wherein the secondcompound is administered for a period of from about three months toabout three years.

[0070] Optionally the administration of the second compound is followedby administration of the first compound for a period of from about threemonths to about three years without the administration of the secondcompound during the period of from about three months to about threeyears.

[0071] Alternatively, the administration of the second compound isfollowed by administration of the first compound for a period greaterthan about three years without the administration of the second compoundduring the greater than about three year period.

[0072] Another aspect of this invention is a synergistic pharmaceuticalcomposition comprising

[0073] a. an amount of a first compound, said first compound beingdroloxifene, raloxifene, tamoxifen or idoxifene; and

[0074] b. an amount of a second compound, said second compound beingsodium fluoride orN-[1(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677

[0075] wherein the amount of the first compound alone and the amount ofthe second compound alone is insufficient to achieve the therapeuticeffects of increase in bone formation and decrease in bone resorption ifadministered simultaneously and wherein the combined effect of theamounts of the first and second compounds is greater than the sum of thetherapeutic effects achievable with the individual amounts of the firstand second compound, and a pharmaceutically acceptable diluent orcarrier.

[0076] A preferred aspect of this synergistic composition is wherein thefirst compound is droloxifene.

[0077] Yet another aspect of this invention is a synergistic method fortreating mammals which present with low bone mass comprisingadministering to a mammal having a condition which presents with lowbone mass

[0078] a. an amount of a first compound, said first compound beingdroloxifene, raloxifene, tamoxifen or idoxifene; and

[0079] b. an amount of a second compound, said second compound beingsodium fluoride orN-[1(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677

[0080] wherein the amount of the first compound alone and the amount ofthe second compound alone is insufficient to achieve the therapeuticeffects of increase in bone formation and decrease in bone resorption ifadministered simultaneously and wherein the combined effect of theamounts of the first and second compounds is greater than the sum of thetherapeutic effects achievable with the individual amounts of the firstand second compound, and a pharmaceutically acceptable diluent orcarrier.

[0081] A preferred aspect of this synergistic method is wherein thefirst compound is droloxifene.

[0082] Another aspect of this invention is a kit containing a treatmentfor a condition which presents with low bone mass comprising:

[0083] a. a therapeutically effective amount of droloxifene, raloxifene,tamoxifen or idoxifene and a pharmaceutically acceptable carrier in afirst unit dosage form;

[0084] b. a therapeutically effective amount of sodium fluoride or N-[1-(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2amino-2-methylpropanamide:MK-677and a pharmaceutically acceptable carrier in a second unit dosage form;and

[0085] c. container means for containing said first and second dosageforms.

[0086] A preferred aspect of this kit is wherein the first compound isdroloxifene.

[0087] Yet another aspect of this invention is a pharmaceuticalcomposition comprising:

[0088] a. a therapeutically effective amount of a first compound, saidfirst compound being

[0089]Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0090](−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0091]Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0092]Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;

[0093]1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0094]Cis-6-(4-hydroxyphenyl)-[4-(2-piperidin-1-yl-ethoxyyphenyl]-5,6,7,8-tetrahydronaphthalene2-ol;or

[0095]1-(4′-Pyrrolidinolethoxyphenyl)-2-phenylthydroxy-1,2,3,4-tetrahydroisoquinoline;and

[0096] b. a therapeutically effective amount of a second compound, saidsecond compound being sodium fluoride, a parathyroid hormone, growthhormone or a growth hormone secretagogue.

[0097] Yet another aspect of this invention is a method for treatingmammals which present with low bone mass comprising administering to amammal having a condition which presents with low bone mass

[0098] a. a therapeutically effective amount of a first compound, saidfirst compound being

[0099]Cis-6-(4-fluoro-phenyl)-5-[4(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0100](−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0101]Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene2-ol;

[0102]Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;

[0103]1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0104]Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;or

[0105]1-(4-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;and

[0106] b. a therapeutically effective amount of a second compound, saidsecond compound being sodium fluoride, a parathyroid hormone, growthhormone or a growth hormone secretagogue.

[0107] A preferred aspect of this method is wherein the condition whichpresents with low bone mass is osteoporosis.

[0108] Another preferred aspect of this method is wherein the firstcompound and the second compound are administered substantiallysimultaneously.

[0109] Another preferred aspect of this method is wherein the secondcompound is administered for a period of from about three months toabout three years.

[0110] Optionally the administration of the second compound is followedby administration of the first compound for a period of from about threemonths to about three years without the administration of the secondcompound during the period of from about three months to about threeyears.

[0111] Alternatively, the administration of the second compound isfollowed by administration of the first compound for a period greaterthan about three years without the administration of the second compoundduring the greater than about three year period.

[0112] Yet another aspect of this invention is a synergisticpharmaceutical composition comprising

[0113] a. an amount of a first compound, said first compound being

[0114]Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0115](−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0116]Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene2-ol;

[0117]Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;

[0118]1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0119]Cis-6-(hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;or

[0120]1-(4′-Pyrrolidinolethoxyphenyl)-2-phenylthydroxy-1,2,3,4-tetrahydroisoquinoline;

[0121] b. an amount of a second compound, said second compound beingsodium fluoride, parathyroid hormone, growth hormone or a growth hormonesecretagogues

[0122] wherein the amount of the first compound alone and the amount ofthe second compound alone is insufficient to achieve the therapeuticeffects of increase in bone formation and decrease in bone resorption ifadministered simultaneously and wherein the combined effect of theamounts of the first and second compounds is greater than the sum of thetherapeutic effects achievable with the individual amounts of the firstand second compound, and a pharmaceutically acceptable diluent orcarrier.

[0123] Yet another aspect of this invention is a synergistic method fortreating mammals which present with low bone mass comprisingadministering to a mammal having a condition which presents with lowbone mass

[0124] a. an amount of a first compound, said first compound being

[0125]Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0126](−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0127]Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene2-ol;

[0128]Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;

[0129]1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0130] Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8tetrahydronaphthalene-2-ol; or

[0131]1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;and

[0132] b. an amount of a second compound, said second compound beingsodium fluoride, parathyroid hormone, growth hormone or a growth hormonesecretagogue

[0133] wherein the amount of the first compound alone and the amount ofthe second compound alone is insufficient to achieve the therapeuticeffects of increase in bone formation and decrease in bone resorption ifadministered simultaneously and wherein the combined effect of theamounts of the first and second compounds is greater than the sum of thetherapeutic effects achievable with the individual amounts of the firstand second compound, and a pharmaceutically acceptable diluent orcarrier.

[0134] Yet another aspect of this invention is a kit containing atreatment for a condition which presents with low bone mass comprising:

[0135] a. a therapeutically effective amount of

[0136]Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0137](−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0138]Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0139]Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;

[0140]1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0141]Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;or

[0142]1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline

[0143] and a pharmaceutically acceptable carrier in a first unit dosageform;

[0144] b. a therapeutically effective amount of sodium fluoride,parathyroid hormone, growth hormone or a growth hormone secretagogue anda pharmaceutically acceptable carrier in a second unit dosage form; and

[0145] c. container means for containing said first and second dosageforms.

[0146] Yet another aspect of this invention is a pharmaceuticalcomposition comprising:

[0147] a. a therapeutically effective amount of a first compound, saidfirst compound being raloxifene, tamoxifen or idoxifene, and

[0148] b. a therapeutically effective amount of a second compound, saidsecond compound being a parathyroid hormone, growth hormone or a growthhormone secretagogue.

[0149] Yet other aspects of this invention are methods of treatmentsynergistic compositions and kits of the immediately precedingcomposition.

[0150] Those skilled in the art will recognize that otheranti-resorptive agents (bisphosphonate, estrogen, estradiol, premarin,eston, estriol or 17α- or 17β-ethynyl estradiol) and other bone anabolicagents (androgen, androgen agonist/antagonist) may be used together orwith any of the agents described herein in this invention in ananalogous manner.

[0151] For example, the anti-resorptive agent droloxifene may becombined with an individual bone anabolic agent such as parathyroidhormone, growth hormone or growth hormone secretagogues.

[0152] The phrase “condition which presents with low bone mass” refersto a condition where the level of bone mass is below the age specificnormal as defined in standards by the World Health Organization“Assessment of Fracture Risk and its Application to Screening forPostmenopausal Osteoporosis (1994), Report of a World HealthOrganization Study Group. World Health Organization TechnicalSeries-843”. Childhood idiopathic and primary osteoporosis are alsoincluded. Included in the treatment of osteoporosis is the prevention orattenuation of long term complications such as curvature of the spine,loss of height, prosthetic surgery, and prevention of prostatemalfunctioning. Also included is increasing the bone fracture healingrate and enhancing the rate of successful bone grafts. Also included isperiodontal disease and alveolar bone loss.

[0153] The phrase “condition which presents with low bone mass” alsorefers to a mammal known to have a significantly higher than averagechance of developing such diseases as are described above includingosteoporosis (e.g., post-menopausal women, men over the age of 60, andpersons being treated with drugs known to cause osteoporosis as a sideeffect (such as glucocorticoid)).

[0154] Those skilled in the art will recognize that the term bone massactually refers to one mass per unit area which is sometimes (althoughnot strictly correctly) referred to as bone mineral density.

[0155] The term “treating”, “treat” or “treatment” as used hereinincludes preventative e.g., prophylactic) and palliative treatment.

[0156] By halo is meant chloro, bromo, iodo, or fluoro.

[0157] By alkyl is meant straight chain or branched saturatedhydrocarbon. Exemplary of such alkyl groups (assuming the designatedlength encompasses the particular example) are methyl, ethyl, propyl,isopropyl, butyl, seo-butyl, tertiary butyl, pentyl, isopentyl, hexyland isohexyl.

[0158] By alkoxy is meant straight chain or branched saturated alkylbonded through an oxy. Exemplary of such alkoxy groups (assuming thedesignated length encompasses the particular example) are methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy,pentoxy, isopentoxy, hexoxy and isohexoxy.

[0159] The expression “pharmaceutically-acceptable anionic salt” refersto nontoxic anionic salts containing anions such as (but not limited to)chloride, bromide, iodide, sulfate, bisuifate, phosphate, acetate,maleate, fumarate, oxalate, lactate, tartrate, citrate, gluconate,methanesulfonate and 4-toluene-sulfonate.

[0160] The expression “pharmaceutically-acceptable cationic salt” refersto nontoxic cationic salts such as (but not limited to) sodium,potassium, calcium, magnesium, ammonium or protonated benzathine(N,N′-dibenzylethylenediamine), choline, ethanolamine, diethanolamine,ethylenediamine, meglamine (N-methylglucamine), benethamine(N-benzylphenethylamine), piperazine or tromethamine(2-amino-2-hydroxymethyl-1,3-propanediol).

[0161] The parenthetical negative or positive sign used herein in thenomenclature denotes the direction plane polarized light is rotated bythe particular stereoisomer.

[0162] As used herein, the expressions “reaction-inert solvent” and“inert solvent” refers to a solvent which does not interact withstarting materials, reagents, intermediates or products in a mannerwhich adversely affects the yield of the desired product.

[0163] The chemist of ordinary skill will recognize that certaincompounds of this invention will contain one or more atoms which may bein a particular stereochemical or geometric configuration, giving riseto stereoisomers and configurational isomers. All such isomers andmixtures thereof are included in this invention. Hydrates of thecompounds of this invention are also included.

[0164] The chemist of ordinary skill will recognize that certaincombinations of heteroatom-containing substituents listed in thisinvention define compounds which will be less stable under physiologicalconditions (e.g. those containing acetal or animal linkages).Accordingly, such compounds are less preferred.

[0165] The pharmaceutical compositions of this invention result in amore rapid and higher magnitude bone mass gain than is achievable withthe same doses of estrogen agonists/antagonists as described above aloneor an agent which stimulates an increase in bone mineral density asdescribed above alone. Thus, these combinations have a synergisticaction, increasing bone mass and decreasing fracture rates to a greaterextent than is achievable through use of either agent alone. Thisinvention makes a significant contribution to the art by providingcompositions and methods that increase and maintain bone mass resultingin prevention, retardation, and/or regression of osteoporosis andrelated bone disorders.

[0166] Other features and advantages will be apparent from thespecification and claims which describe the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0167] The first compound of this invention is a mammalian estrogenagonist/antagonist. Any estrogen agonist/antagonist may be used as thefirst compound of this invention. The term estrogen agonist/antagonistrefers to compounds which bind with the estrogen receptor, inhibit boneturnover and prevent bone loss. Such activities are readily determinedby those skilled in the art according to standard assays includingestrogen receptor binding assays (see in Vitro Estrogen Receptor BindingAssay hereinafter), standard bone histomorphometric and densitometermethods (see Estrogen Agonist/Antagonist Protocol hereinafter, andEriksen E. F. et al., Bone Histomorphometry, Raven Press, New York,1994, pages 1-74; Grier S. J. et. al., The Use of Dual-Energy X-RayAbsorptiometry In Animals, Inv. Radiol., 1996,31(1):50-62; Wahner H. W.and Fogelman I., The Evaluation of Osteoporosis: Dual Energy X-RayAbsorptiometry in Clinical Practice., Martin Dunitz Ltd., London 1994,pages 1-296). A variety of these compounds are described and referencedbelow, however, other estrogen agonists/antagonists will be known tothose skilled in the art.

[0168] A preferred estrogen agonist/antagonist is droloxifene: (phenol,3-[1-[4[2-(dimethylamino)ethoxy] phenyl]-2-phenyl-1-butenyl]-, (E)-) andassociated compounds which are disclosed in U.S. Pat. No. 5,047,431 (thedisclosure of which is hereby incorporated by reference).

[0169] Another preferred estrogen agonist/antagonist is tamoxifen:(ethanamine,2-[ 4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethyl, (Z)-2-,2-hydroxy-1,2,3-propanetricarboxylate(1:1)) and associated compoundswhich are disclosed in U.S. Pat. No. 4,536,516 (the disclosure of whichis hereby incorporated by reference). Another related compound is4-hydroxy tamoxifen which is disclosed in U.S. Pat. No. 4,623,660 (thedisclosure of which is hereby incorporated by reference).

[0170] Another preferred estrogen agonist/antagonist is raloxifene:(methanone,-[6-hydroxy-2-(4-hydroxyphenyl)benzo-[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl].,hydrochloride) and associated compounds which are disclosed In U.S.Pat. No. 4,418,068 (the disclosure of which is hereby incorporated byreference).

[0171] Another preferred estrogen agonist/antagonist is toremifene:(ethanamine, 2-[4-(4chloro-1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethyl-, (Z)-,2-hydroxy-1,2,3-propanetricarboxylate (1:1) and associated compoundswhich are disclosed in U.S. Pat. No. 4,996,225 (the disclosure of whichis hereby incorporated by reference).

[0172] Another preferred estrogen agonist/antagonist is centchroman:1-[2-[[4-(-methoxy-2,2-dimethyl-3-phenyl-chroman-4-yl)-phenoxy]-ethyl]-pyrrolidine,and associated compounds which are disclosed in U.S. Pat. No. 3,822,287(the disclosure of which is hereby incorporated by reference).

[0173] Another preferred estrogen agonist/antagonist is idoxifene:Pyrrolidine, 1-[-[4-[[1-(4-odophenyl)-2-phenyl-1-Butenyl]phenoxy]ethyl]and associated compounds which are disclosed in U.S. Pat. No. 4,839,155(the disclosure of which is hereby incorporated by reference).

[0174] Other preferred estrogen agonist/antagonists include compounds ofthe formula

[0175] wherein:

[0176] A is selected from CH₂and NR;

[0177] B, D and E are independently selected from CH and N;

[0178] Y is

[0179] (a) phenyl, optionally substituted with 1-3 substituentsindependently selected from R⁴;

[0180] (b) naphthyl, optionally substituted with 1-3-substituentsindependently selected from R⁴;

[0181] (c) C₃-C₈ cycloalkyl, optionally substituted with 1-2substituents independently selected from R⁴;

[0182] (d) C₃-C₈ cycloalkenyl, optionally substituted with 1-2substituents independently selected from R⁴;

[0183] (e) a five membered heterocycle containing up to two heteroatomsselected from the group consisting of —O—, —NR²— and —S(O)_(n)—,optionally substituted with 1-3 substituents independently selected fromR⁴;

[0184] (f) a six membered heterocycle containing up to two heteroatomsselected from the group consisting of —O—, —NR²— and —S(O)_(n)—optionally substituted with 1-3 substituents independently selected fromR⁴; or

[0185] (g) a bicyclic ring system consisting of a five or six memberedheterocyclic ring fused to a phenyl ring, said heterocyclic ringcontaining up to two heteroatoms selected from the group consisting of—O—, —NR²— and —S(O)_(n)—, optionally substituted with 1-3substituents-independently-selected from R⁴;

[0186] Z¹ is

[0187] (a) —(CH₂)_(p) W(CH₂)_(q)—;

[0188] (b) —O(CH₂)_(p) CR⁵R⁸—;

[0189] (c) —O(CH₂)_(p)W(CH₂)_(q);

[0190] (d) —OCHR²CHR³—; or

[0191] (e) —SCHR²CHR³—;

[0192] G is

[0193] (a) —NR⁷R⁸;

[0194] (b)

[0195] wherein n is 0, 1 or 2; m is 1, 2 or 3; Z² is —NH—, —O—, —S—, or—CH₂—; optionally fused on adjacent carbon atoms with one or two phenylrings and, optionally independently substituted on carbon with one tothree substituents and, optionally, independently on nitrogen with achemically suitable substituent selected from R⁴; or

[0196] (c) a bicyclic amine containing five to twelve carbon atoms,either bridged or fused and optionally substituted with 1-3 substituentsindependently selected from R⁴; or

[0197] Z² and G in combination may be

[0198] W is

[0199] (a) —CH₂—;

[0200] (b) —CH═CH—;

[0201] (c) —O—;

[0202] (d) —NR²—;

[0203] (e) —S(O)_(n)—;

[0204] (f)

[0205] (g) —CR²(OH)—;

[0206] (h) —CONR²—;

[0207] (i) —NR²CO—;

[0208] (j)

[0209] (k) —C≡C—;

[0210] R is hydrogen or C₁-C₈ alkyl;

[0211] R² and R³ are independently

[0212] (a) hydrogen; or

[0213] (b) C₁-C₄ alkyl;

[0214] R⁴ is

[0215] (a) hydrogen;

[0216] (b) halogen;

[0217] (c) C₁-C₈ alkyl;

[0218] (d) C₁-C₄ alkoxy;

[0219] (e) C₁-C₄ acyloxy;

[0220] (f) C₁-C₄ alkylthio;

[0221] (g) C₁-C₄ alkylsufonyl;

[0222] (h) C₁-C₄ alkylsulfonyl;

[0223] (i) hydroxy (C₁-C₄)alkyl;

[0224] (j) aryl (C₁-C₄)alkyl;

[0225] (k) —CO₂H;

[0226] (l) —CN;

[0227] (m) —CONHOR;

[0228] (n) —SO₂NHR;

[0229] (o) —NH₂;

[0230] (p) C₁-C₄ alkyl amino;

[0231] (q) C₁-C₄ dialkylamino;

[0232] (r) —NHSO₂R;

[0233] (s) —NO₂;

[0234] (t) -aryl; or

[0235] (u) —OH;

[0236] R⁵ and R⁸ are independently C₁-C₈ alkyl or together form a C₃-C₁₀carbocyclic ring;

[0237] R⁷ and R⁸ are independently

[0238] (a) phenyl;

[0239] (b) a C₃-C₁₀ carbocyclic ring, saturated or unsaturated;

[0240] (c) a C₃-C₁₀ heterocyclic ring containing up to two heteroatoms,selected from —O—, —N— and —S—;

[0241] (d) H;

[0242] (e) C₁-C₈ alkyl; or

[0243] (f) form a 3 to 8 membered nitrogen containing ring with R⁵ orR⁸;

[0244] R⁷ and R⁸ in either linear or ring form may optionally besubstituted with up to three substituents independently selected fromC₁-C₈ alkyl, halogen, alkoxy, hydroxy and carboxy;

[0245] a ring formed by R⁷ and R⁸ may be optionally fused to a phenylring;

[0246] e is 0, 1 or 2;

[0247] m is 1, 2 or 3;

[0248] n is 0, 1 or 2;

[0249] p is 0, 1, 2 or 3;

[0250] q is 0, 1, 2 or 3;

[0251] and optical and geometric isomers thereof; and nontoxicpharmacologically acceptable acid addition salts, N-oxides, esters andquaternary ammonium salts thereof.

[0252] Preferred compounds of the invention are of the formula:

[0253] wherein G is

[0254] R⁴ is H, OH, F, or Cl; and B and E are independently selectedfrom CH and N.

[0255] Especially preferred compounds are:

[0256]Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0257](−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0258]Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthaJene-2-ol;

[0259]Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;

[0260]1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0261]Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;and

[0262]1-(4′-Pyrrolidinolethoxyphenyl)-2-phenylhydroxy-1,2,3,4tetrahydroisoquinoline.

[0263] The above compounds of this invention are readily prepared by thereactions illustrated in the schemes below.

[0264] Certain compounds of formula I are conveniently prepared from anunsaturated intermediate

[0265] by hydrogenation with a noble metal catalyst in a reaction inertsolvent. Pressure and temperatures are not critical and hydrogenation isnormally accomplished in a few hours at room temperatures at 20-80 psihydrogen pressure.

[0266] The hydrogenated product is isolated, purified if desired, andthe ether group is cleaved with an acidic catalyst in a reaction inertsolvent at a temperature between 0° C. to 100° C. depending on theacidic catalyst used. Hydrogen bromide at elevated temperatures, borontribromide and aluminum chloride at 0° C. to ambient temperature havebeen found to be effective for this reaction.

[0267] The product, Formula I is isolated and purified by standardprocedures.

[0268] Intermediates of Formula Il where A is CH₂, and B, D and E are CHare described in U.S. Pat. No. 3,274,213; J. Med. Chem 10, 78 (1967); J.Med. Chem 10, 138 (1967); and J. Med. Chem. 12, 881 (1969), thedisclosures of which are herein incorporated by reference. They can alsobe prepared by procedures described below.

[0269] The preparation of the compounds of Formula I where e=1, A═CH₂,Z¹═OCH₂CH₂, G=cycloalkylamine, B═CH is shown in Scheme 1. Compounds 1-2,where D and E are CH are made by alkylation of 4-bromophenol with thecorresponding N-chloroethytamine using potassium carbonate as base in apolar aprotic solvent like dimethylformamide at elevated temperatures. Apreferred temperature is 100° C. Compounds 1-2 where D or E or both areN are synthesized using a nucleophilic displacement reaction performedon dibromides (1-1) using hydroxy ethyl cycloalkylamines under phasetransfer conditions to afford bromo amines (1-2). Synthesis, 77, 673(1980). Following halogen metal exchange using n-butyllithium ormagnesium metal, bromo amines (1-2) yield the corresponding lithium ormagnesium reagents which are allowed to react at low temperature in thepresence of cesium chloride preferably (without cesium chloride thereaction also proceeds) with 6-methoxy-1-tetralone to afford eithercarbinols (1-3) or styrenes (1-4) after acidic workup. Treatment ofeither carbinols (1-3) or styrenes (1-4) with a brominating agent suchas pyridinium bromide perbromide affords bromo styrenes (1-5). Aryl orheteroaryl zinc chlorides or aryl or heteroaryl boronic acids react withbromides (1-5) in the presence of a palladium metal catalyst liketetrakis triphenyl phosphine palladium (O) to yield diaryl styrenes(1-6). [Pure & Applied Chem. 63, 419,(1991) and Bull. Chem. Soc. Jpn.61, 3008-3010, (1988)] To prepare the preferred compounds thesubstituted phenyl zinc chlorides or substituted phenylboronic acids areused in this reaction. The aryl zinc chlorides are prepared by quench ofthe corresponding rithium reagent with anhydrous zinc chloride. The arylboronic acids, that are not commercially available, are prepared byquenching the corresponding aryl lithium reagent with trialkyl borate,preferably the trimethyl or triisopropyl borate, followed by aqueousacid workup. Acta Chemica Scan. 47, 221-230 (1993). The lithium reagentsthat are not commercially available are prepared by halogen metalexchange of the corresponding bromide or halide with n-butyl ort-butyllithium. Alternately, the lithium reagent is prepared byheteroatom facilitated lithiations as described in Organic Reactions,Volume 27, Chapter 1. Catalytic hydrogenation of 1-6 in the presence ofpalladium hydroxide on charcoal, for example, affords the correspondingdihydro methoxy intermediates which were subsequently demethylated usingboron tribromide at 0° C. in methylene chloride or 48% hydrogen bromidein acetic acid at 80-100° C. to afford target structures (1-7). Thesecompounds are racemic and can be resolved into the enantiomers via highpressure liquid chromatography using a column with a chiral stationaryphase like the Chiralcel OD columns. Alternately optical resolution canbe carried out by recrystallization of the diasereomeric salts formedwith optically pure acids like 1,1′-binapthyl-2,2′-diyl hydrogenphosphate.

[0270] The cis compounds (1-7) can be isomerized to the trans compoundson treatment with base.

[0271] When D and/or E is nitrogen the intermediates (Formula II) andcompounds of Formula I may be prepared from the correspondingdihalopyridines or pyrimidines as illustrated in Scheme 1.

[0272] The methyl ether of the compound of Formula I where e=1, A═CH₂,Z¹═OCH₂CH₂, G=pyrrolidine, D,E, B═CH, Y═Ph can also be convenientlyprepared by a first step of hydrogenation of nafoxidine (Upjohn & Co.,700 Portage Road, Kalamazoo, Miss. 49001) in a reaction inert solvent inthe presence of a nobel metal catalyst. Pressure and temperature are notcrtical; the reaction is conveniently run in ethanol at room temperaturefor approximately 20 hours at 50 psi.

[0273] The second step is cleavage of the methoxy group which isaccomplished conveniently at room temperature with an acidic catalystsuch as boron tribromide in a reaction inert solvent or at 80-100° C.with hydrogen bromide in acetic acid. The product is then isolated byconventional methods and converted to an acid salt if desired.

[0274] Compounds of formula I wherein B is nitrogen are prepared by theprocedures illustrated in Scheme 2 and 3.

[0275] The synthesis of compounds of Formula I where B═N is shown inScheme 2. Aryl acid chlorides (2-1) on treatment with primary aminesafford aryl secondary amides (2-2), which are reduced with lithiumaluminum hydride in ethereal solvents to yield secondary amines (2-3).Subsequent acylation of (2-3) with aroyl acid chlorides leads totertiary amides (2-4), which are cyclized in hot phosphorus oxychlorideto yield dihydro isoquinolinium salts (2-5). Reduction with sodiumborohydride to alkoxytetrahydro isoquinolines: followed by borontribromide demethylation in methylene chloride affords the targetstructures.

[0276] The synthesis of the compounds of Formula I where B═N is alsodescribed below in Scheme 3. Secondary amines (3-1) on acylation withbenzyloxyaroyl chlorides (3-2) afford tertiary amides (3-3) which oncyclization with hot phosphorous oxychloride yield dihydro isoquinolinesalts (3-4). Sodium borohydride reduction of (3-4) followed bydebenzylation with aqueous hydrochloric acid affords isoquinolines(3-5), which are alkylated with the appropriately functionalizedchlorides and dimethylated with boron tribromide to yield the desiredtarget structures.

[0277] Other estrogen agonist/antagonists are described in U.S. Pat. No.4,133,814 (the disclosure of which is hereby incorporated by reference).U.S. Pat. No. 4,133,814 discloses derivatives of2-phenyl-3-aroyl-benzothiophene and2-phenyl-3-aroylbenzothiophene-1-oxide.

[0278] Lednicer, et al., J. Med. Chem., 12, 881 (1969) describe estrogenantagonists of the structure

[0279] wherein R² is phenyl or cyclopentyl and R³ is H,

[0280] U.S. Pat. No. 3,234,090 (the disclosure of which is herebyincorporated by reference) discloses estrogen agonist/antagonist offormula

[0281] in which Ph is a 1,2-phenylene radical, Ar is a monocycliccarbocyclic aryl group substituted by tertiary amino-lower alkyl-oxy, inwhich tertiary amino is separated from oxy by at least two carbon atoms,R is hydrogen, an aliphatic radical, a carbocyclic aryl radical, acarbocyclic aryl-aliphatic radical, a heterocyclic aryl radical or aheterocyclic aryl aliphatic radical, the group of the formula-(C_(n)H_(2n−2))- stands for an unbranched alkylene radical having fromthree to five carbon atoms and carrying the groups Ar and R₁ salts,N-oxides, salts of N-oxides or quaternary ammonium compounds thereof, aswell as a procedure for the preparation of such compounds.

[0282] U.S. Pat. No. 3,277,106 (the disclosure of which is herebyincorporated by reference) discloses basic ethers with estrogenagonist/antagonist effects which are of the formula

[0283] in which Ph is a 1,2-phenylene radical, Ar is a monocyclic arylradical substituted by at least one amino-lower alkyl-oxy group in whichthe nitrogen atom is separated from the oxygen atom by at least twocarbon atoms, R is an aryl radical, and the portion -(C_(n)H_(2n−2))-stands for lower alkylene forming with Ph a six- or seven-membered ring,two of the ring carbon atoms thereof carry the groups Ar and R, salts,N-oxides, salts of N-oxides and quaternary ammonium compounds thereof.

[0284] U.S. Pat. No. 3,274,213 (the disclosure of which is herebyincorporated by reference) discloses estrogen agonist/antagonistcompounds of the formula

[0285] wherein R₁ and R₂ are selected from the class consisting of loweralkyl and lower alkyl linked together to form a 5 to 7 ring membersaturated heterocyclic radical.

[0286] The second compound of this invention may be any compound asdescribed below that augments bone mass to a level which is above thebone fracture threshold (as detailed in the World Health OrganizationStudy World Health Organization, “Assessment of Fracture Risk and itsApplication to Screening for Postmenopausal Osteoporosis (1994). Reportof a WHO Study Group. World Health Organization Technical Series 843”).

[0287] Any prostaglandin, or prostaglandin agonist/antagonist may beused as the second compound of this invention. Those skilled in the atwill recognize that sodium fluoride, parathyroid hormone (PTH), activefragments of parathyrold hormone, growth hormone or growth hormonesecretagogues may also be used. The following paragraphs describeexemplary second compounds of this invention in greater detail.

[0288] Any prostaglandin may be used as the second compound of thisinvention. The term prostaglandin refers to compounds which are analogsof the natural prostaglandins PGD₁, PGD₂, PGE₂, PGE₁ and PGF₂α which areuseful in the treatment of osteoporosis. These compounds bind to theprostaglandin receptors. Such binding is readily determined by thoseskilled in the art according to standard assays (e.g., An S. et al.,Cloning and Expression of the EP₂ Subtype of Human Receptors forProstaglandin E₂, Biochemical and Biophysical Research Communications,1993, 197(1):263-270).

[0289] Prostaglandins are alicyclic compounds related to the basiccompound prostanoic acid. The carbon atoms of the basic prostaglandinare numbered sequentially from the carboxylic carbon atom through thecyclopentyl ring to the terminal carbon atom on the adjacent side chain.Normally the adjacent side chains are in the trans orientation. Thepresence of an oxo group at C-9 of the cyclopentyl moiety is indicativeof a prostaglandin within the E class while PGE₂ contains a transunsaturated double bond at the C₁₃-C₁₄ and a cis double bond at theC₅-C₈ position.

[0290] A variety of prostaglandins are described and referenced below,however, other prostaglandins will be known to those skilled in the art.Exemplary prostaglandins are disclosed in U.S. Pat. Nos. 4,171,331 and3,927,197 (the disclosures of which we hereby incorporated byreference).

[0291] Norrdin et al., The Role of Prostaglandins in Bone In Vivo,Prostaglandins Leukotriene Essential Fatty Acids 41, 139-150, 1990 is areview of bone active prostaglandins.

[0292] Any prostaglandin agonist/antagonist may be used as the secondcompound of this invention. The term prostaglandin agonist/antagonistrefers to compounds which bind to prostaglandin receptors (e.g., An S.et al., Cloning and Expression of the EP₂ Subtype of Human Receptors forProstaglandin E₂, Biochemical and Biophysical Research Communications,1993, 197(1):263-270) and mimic the action of prostaglandin in vivo(e.g., stimulate bone formation and increase bone mass). Such actionsare readily determined by those skilled in the art according to standardassays (e.g., see Anabolic Agent Protocol described hereinafter andErksen E. F. et al., Bone Histomorphometry, Raven Press, New York, 1994,pages 1-74; Grier S. J. et. al., The Use of Dual-Energy X-RayAbsorptiometry In Animals, Inv. Radiol., 1996, 31(1):50-62; Wahner H. W.and Fogelman I., The Evaluation of Osteoporosis: Dual Energy X-RayAbsorptiometry In Clinical Practice., Martin Dunitz Ltd., London 1994,pages 1-296). A variety of these compounds are described and referencedbelow, however, other prostaglandin agonists/antagonists will be knownto those skilled in the art. Exemplary prostaglandinagonists/antagonists are disclosed as follows.

[0293] Commonly assigned U.S. Pat. No. 3,932,389 (the disdosure of whichis hereby incorporated by reference) discloses2-descarboxy-2-(tetrazol-5-yl)-11-desoxy-15-substituted-omega-pentanorprostaglandinsuseful for bone formation activity.

[0294] Commonly assigned U.S. Pat. No. 4,018,892 (the disclosure ofwhich is hereby incorporated by reference) discloses16-aryl-13,14-dihydro-PGE₂ p-biphenyl esters useful for bone formationactivity.

[0295] Commonly assigned U.S. Pat. No. 4,219,483 (the disclosure ofwhich is hereby incorporated by reference)discloses-2,3,6-substituted-4-pyrones useful for bone formationactivity.

[0296] Commonly assigned U.S. Pat. No. 4,132,847 (the disclosure ofwhich is hereby incorporated by reference) discloses2,3,6-substituted-4-pyrones useful for bone formation activity.

[0297] U.S. Pat. No. 4,000,309 (the disclosure of which is herebyincorporated by reference) discloses 16-aryl-13,14-dihydro-PGE₂p-biphenyl esters useful for bone formation activity.

[0298] U.S. Pat. No. 3,982,016 (the disclosure of which is herebyincorporated by reference) discloses 16-aryl-13,14-dihydro-PGE₂p-biphenyl esters useful for bone formation activity.

[0299] U.S. Pat. No. 4,621,100 (the disclosure of which is herebyincorporated by reference) discloses substituted cyclopentanes usefulfor bone formation activity.

[0300] U.S. Pat. No. 5,216,183 (the disclosure of which is herebyincorporated by reference) discloses cyclopentanones useful for boneformation activity.

[0301] Sodium fluoride may be used as the second compound of thisinvention. The term sodium fluoride refers to sodium fluoride in all itsforms (e.g., slow release sodium fluoride, sustained release sodiumfluoride). Sustained release sodium fluoride is disclosed in U.S. Pat.No. 4,904,478, the disclosure of which is hereby incorporated byreference. The activity of sodium fluoride is readily determined bythose skilled in the art according to biological protocols (e.g., seeAnabolic Agent Protocol described hereinafter and Eriksen E. F. et al.,Bone Histomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S.J. et. al., The Use of Dual-Energy X-Ray Absorptiometry in Animals, Inv.Radiol., 1996, 31(1):50-62; Wahner H. W. and Fogelman I., The Evaluationof Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice.,Martin Dunitz Lid., London 1994, pages 1-296).

[0302] Any parathyroid hormone (PTH) may be used as the second compoundof this invention. The term parathyroid hormone refers to parathyroldhormone, fragments or metabolites thereof and structural analogs thereofwhich can stimulate bone formation and increase bone mass. Suchfunctional activity is readily determined by those skilled in the artaccording to standard assays (e.g., see Anabolic Agent Protocoldescribed hereinafter and Eriksen E. F. et al., Bone Histomorphometry,Raven Press, New York, 1994, pages 1-74; Grier S. J. et. al., The Use ofDual-Energy X-Ray Absorptiometry In Animals, Inv. Radiol.,1996,31(1):50-62; Wahner H. W. and Fogelman I., The Evaluation ofOsteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice.,Martin Dunitz Ltd., London 1994, pages 1-296). A variety of thesecompounds are described and referenced below, however, other parathyroidhormones will be known to those skilled in the art. Exemplaryparathyroid hormones are disclosed in the following references.

[0303] “Human Parathyroid Peptide Treatment of Vertebral Osteoporosis”,Osteoporosis Int., 3, (Supp 1):199-203.

[0304] “PTH 1-34 Treatment of Osteoporosis with Added HormoneReplacement Therapy: Biochemical, Kinetic and Histological Responses”Osteoporosis Int. 1:162-170.

[0305] Any growth hormone or growth hormone secretagogue may be used asthe second compound of this invention. The term growth hormonesecretagogue refers to compounds which stimulate the release of growthhormone or mimic the action of growth hormone (e.g., increase boneformation leading to increased bone mass). Such actions are readilydetermined by those skilled in the art according to standard assays(e.g., as described hereinafter). A variety of these compounds areincluded in the following published PCT patent applications WO 95/14666;WO 95/13069; WO 94/19367; WO 94113696; and WO 95/34311. However, othergrowth hormone or growth hormone secretagogues will be known to thoseskilled in the art.

[0306] In particular a preferred growth hormone secretagogue isN-[1-(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidln]-1′-yl)cabonyl]-2-(phenytmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677.

[0307] Other preferred growth hormone secretagogues include

[0308]2-amino-N-[2-(3a-(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-2-oxo-ethyl]-isobutyramideoritsL-tartaric acid salt;

[0309]2-amino-N-{1-(R)-benzyloxymethyl-2-[3a-(R)(4-fluoro-benzyl)-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5yl]-2oxo-ethyl}isobutyramide;and

[0310]2-amino-N-[2-(3a-(R)-benzyl4oxo-2,3,3a,4,6,7-tetrahydro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)benzyloxymethyl-2-oxo-ethyl]isoboide.

[0311] In general, the compounds of this invention can be made byprocesses which include processes known in the chemical arts,particularly in light of the description contained herein.

[0312] Some of the preparation methods useful for making the compoundsof this invention may require protection of remote functionality (i.e.,primary amine, secondary amine, carboxyl). The need for such protectionwill vary depending on the nature of the remote functionality and theconditions of the preparation methods. The need for such protection isreadily determined by one skilled in the art. The use of suchprotection/deprotection methods is also within the skill in the art. Fora general description of protecting groups and their use, see T. W.Greene, Protective Groups in Organic Synthesis. John Wiley & Sons, NewYork, 1991. The starting materials and reagents for the compounds ofthis invention are also readily available or can be easily synthesizedby those skilled in the art using conventional methods of organicsynthesis. For example, many of the compounds used herein are, arerelated to, or are derived from compounds found in nature, in whichthere is a large scientific interest and commercial need, andaccordingly many such compounds are commercially available or arereported in the literature or are easily prepared from other commonlyavailable substances by methods which are reported in the literature.Such compounds include, for example, prostaglandins.

[0313] Some of the compounds of this invention have asymmetric carbonatoms and therefore are enantiomers or diastereomers. Diasteromericmixtures can be separated into their individual diastereomers on thebasis of their physical chemical differences by methods known per se.,for example, by chromatography and/or fractional crystallization.Enantiomers can be separated by converting the enantiomeric mixture intoa diasteromeric mixture by reaction with an appropriate optically activecompound (e.g., alcohol), separating the diastereomers and converting(e.g., hydrolyzing) the individual diastereomers to the correspondingpure enantiomers. All such isomers, including diastereomers, enantiomersand mixtures thereof are considered as part of this invention.

[0314] Although many compounds of this invention are not ionizable atphysiological conditions, some of the compounds of this invention areionizable at physiological conditions. Thus, for example some of thecompounds of this invention are acidic and they form a salt with apharmaceutically acceptable cation. All such salts are within the scopeof this invention and they can be prepared by conventional methods. Forexample, they can be prepared simply by contacting the acidic and basicentities, usually in a stoichiometric ratio, in either an aqueous,non-aqueous or partially aqueous medium, as appropriate. The salts arerecovered either by filtration, by precipitation with a non-solventfollowed by filtration, by evaporation of the solvent, or, in the caseof aqueous solutions, by lyophilization, as appropriate.

[0315] In addition, some of the compounds of this invention are basic,and they form a salt with a pharmaceutically acceptable anion. All suchsalts are within the scope of this invention and they can be prepared byconventional methods. For example, they can be prepared simply bycontacting the acidic and basic entities, usually in a stoichiometricratio, in either an aqueous, non-aqueous or partially aqueous medium, asappropriate. The salts are recovered either by filtration, byprecipitation with a non-solvent followed by filtration, by evaporationof the solvent, or, in the case of aqueous solutions, by lyophilization,as appropriate.

[0316] In addition, when the compounds of this invention form hydratesor solvates they are also within the scope of the invention.

[0317] The pharmaceutical combinations and methods of this invention areall adapted to therapeutic use as agents that either activate boneturnover or prevent bone resorption or increase bone formation inmammals, particularly humans. Since these functions are closely relatedto the development of osteoporosis and bone related disorders, thesecombinations, by virtue of their action on bone, prevent, arrest,regress or reverse osteoporosis.

[0318] The utility of the compounds of the present invention as medicalagents in the treatment of conditions which present with low bone mass(e.g., osteoporosis) in mammals (e.g. humans, particularly the female)is demonstrated by the activity of the compounds of this invention inconventional assays and the in vitro and in vivo assays described below(COMBINATION AND SEQUENTIAL TREATMENT PROTOCOL; ESTROGENAGONIST/ANTAGONIST PROTOCOL; ANABOUC AGENT PROTOCOL; IN VITRO ESTROGENRECEPTOR BINDING ASSAY; AND GROWTH HORMONE/GROWTH HORMONE SECRETAGOGUEPROTOCOL). Such assays also provide a means whereby the activities ofthe compounds of this invention can be compared between themselves andwith the activities of other known compounds. The results of thesecomparisons are useful for determining dosage levels in mammals,including humans, for the treatment of such diseases.

COMBINATION AND SEQUENTIAL TREATMENT PROTOCOL

[0319] The following protocols can of course be varied by those skilledin the art. For example, intact male or female rats, sex hormonedeficient male (orchidectomy) or female (ovariectomy) rats may be used.In addition, male or female rats at different ages (such as 12 months ofage) can be used in the studies. The rats can be either intact orcastrated (ovariectomized ororchidectornized), and administrated withanabolic agents such as prostaglandin E2 (PGE2) at different doses (suchas 1, 3 or 6 mg/kg/day) for a certain period (such as two weeks to twomonths), and followed by administration of an anti-resorptive agent suchas droloxifene at different doses (such as 1,5,10 mg/kg/day) for acertain period (such as two weeks to two months), or a combinationtreatment with both anabolic agent and anti-resorptive agent atdifferent doses for a certain period (such as two weeks to two months).In the castrated rats, treatment can be started at the next day aftersurgery (for the purpose of preventing bone loss) or at the time boneloss has already occurred (for the purpose of restoring bone mass).

[0320] The following protocols are described as using PGE2 as the boneanabolic agent and droloxifene as the antiresorptive agent, however,other anabolic agents and antiresorptive agents may be tested in theprotocol.

[0321] One hundred and four female Sprague-Dawley rats (Charles River,Wilmington, Mass.) at 12 months of age are sham-operated orovariectomized (OVX) at month 0. Three months post-surgery, OVX ratsreceive either Prostaglandin E₂ (PGE₂), a known anabolic bone agent, at3 mg/kg/day (subcutaneously injection), or PGE₂ at 3 mg/kg/day(subcutaneously injection) combined with droloxifene (DRO) at 10mg/kg/day (orally) for 2 months. Thereafter, the PGE₂ treatment iswithdrawn and the rats are treated with either vehicle (10% alcohol insaline) or DRO (10 mg/kg/day, orally) for another one and a half monthsas described in the following.

[0322] Group I: Eight rats are autopsied at month 0 as basal controls

[0323] Group II: Eight sham-operated rats are autopsied at month 3 aspretreatment controls Group III: Eight sham-operated rats are orallytreated with vehicle (10% ethanol in saline) from months 3 to 5, andautopsied at month 5.

[0324] Group IV: Eight sham-operated rats are orally treated withvehicle (10% ethanol in saline) from months 3 to 6.5, and autopsied atmonth 6.5.

[0325] Group V: Eight OVX rats are autopsied at month 3 as pretreatmentcontrols

[0326] Group VI: Eight OVX rats are orally treated with vehicle (10%ethanol in saline) from months 3 to 5, and autopsied at month 5.

[0327] Group VII: Eight OVX rats are orally treated with vehicle (10%ethanol in saline) from months 3 to 6.5, and autopsied at month 6.5.

[0328] Group VIII: Eight OVX rats are subcutaneously injected with 3mg/kg/day of PGE2 from months 3 to 5, and autopsied at month 5.

[0329] Group IX: Eight OVX rats are subcutaneously injected with 3mg/kg/day of PGE2 from months 3 to 5, and vehicle from 5 to 6.5 months,and then autopsied at month 6.5.

[0330] Group X: Eight OVX rats are subcutaneously injected with 3mg/kg/day of PGE2 from months 3 to 5, and 10 mg/kg/day of DRO orallyfrom 5 to 6.5 months, and then autopsied at month 6.5.

[0331] Group Xl: Eight OVX rats are subcutaneously injected with 3mg/kg/day of PGE2 and 10 mg/kg/day of DRO orally from months 3 to 5, andthen autopsied at month 5.

[0332] Group XII: Eight OVX rats are subcutaneously injected with 3mg/kg/day of PGE2 and 10 mg/kg/day of DRO orally from months 3 to 5, andvehicle from months 5 to 6.5, then autopsied at month 6.5.

[0333] Group XIII: Eight OVX rats are subcutaneously injected with 3mg/kg/day of PGE2 and 10 mg/kg/day of DRO orally from months 3 to 5, andDRO alone from months 5 to 6.5, then autopsied at month 6.5.

[0334] Both PGE2 (Cayman Chemical Co., Ann Arbor, Mich.) or droloxifene(Pfizer Inc. Groton, Conn.) powder are first dissolved in 100% ethanoland further diluted with saline into desired concentrations (finalethanol concentration was 10%). PGE solution is daily injectedsubcutaneously on the back at 1 ml/kg. Droloxifene solution is givendaily p.o. at 1 ml/rat. All rats are given subcutaneous injections of 10mg/kg kalcein (fluorochrome bone marker, Sigma Chemical Co. St. LouisMo.) twelve and two days before death to examine the dynamic changes inbone tissues.

[0335] The rats are sacrificed under ketamine anesthesia. The followingendpoints are determined:

[0336] Femoral Bone Mineral Measurements

[0337] The right femur from each rat is removed at autopsy and scannedusing dual energy xray absorptiometry (DXA, QODR 1000/W, Hologic Inc.,Waltham, Mass.) equipped with “Regional High Resolution Scan” software(Hologic Inc., Waltham, Mass.). The scan field size is 5.08×1.902 cm,resolution is 0.0254×0.0127 cm and scan speed is 7.25 mm/second. Thefemoral scan images are analyzed and bone area, bone mineral content(BMC), and bone mineral density (BMD) of whole femora (WF), distalfemoral metaphyses (DFM), femoral shaft (FS), and proximal femora (PF)are determined.

[0338] LumbarVertebral Bone Mineral Measurements

[0339] Dual energy xray absorptiometry (QDR 1000/W, Hologic, Inc.,Waltham, Mass.) equipped with a “Regional High Resolution Scan” software(Hologic, Inc., Waltham, Mass.) is used to determined the bone area,bone mineral content (BMC), and bone mineral density (BMD) of wholelumbar spine and each of the six lumbar vertebrae (LV1-6) in theanesthetized rats. The rats are anesthetized by injection (i.p.) of 1ml/kg of a mixture of ketamine/rompun (ratio of 4 to 3), and then placedon the rat platform. The scan field sized is 6×1.9 cm, resolution is0.0254×0.0127 cm, and scan speed is 7.25 mm/sec. The whole lumbar spinescan image is obtained and analyzed. Bone area (BA), and bone mineralcontent (BMC) is determined, and bone mineral density is calculated (MBCdivided by BA) for the whole lumbar spine and each of the six lumbarvertebrae (LV1-6).

[0340] Proximal Tibial Metaphvseal Cancellous Bone HistomorphometricAnalyses

[0341] The right tibia is removed at autopsy, dissected free of muscle,and cut into three parts. The proximal tibia is fixed in 70% ethanol,dehydrated in graded concentrations of ethanol, defatted in acetone,then embedded in methyl methacrylate (Eastman Organic Chemicals,Rochester, N.Y.). Frontal sections of proximal tibial metaphyses at 4and 10 μm thickness is cut using Reichert-Jung Polycut S microtome. One4 μm and one 10 μm sections from each rat is used for cancellous bonehistomorphometry. The 4 μm sections is stained with modified Masson'sTrichrome stain while the 10 μm sections remained unstained.

[0342] A Bioquant OS/2 histomorphometry system (R&M biometrics, Inc.,Nashville, Tenn.) is used for the static and dynamic histomorphometricmeasurements of the secondary spongiosa of the proximal tibialmetaphyses between 1.2 and 3.6 mm distal to the growth plate-epiphysealjunction. The first 1.2 mm of the tibial metaphyseal region needs to beomitted in order to restrict measurements to the secondary spongiosa.The 4 μm sections are used to determine indices related to bone volume,bone structure, and bone resorption, while the 10 μm sections are usedto determine indices related to bone formation and bone turnover.

[0343] I. Measurements and calculations related to trabecular bonevolume and structure

[0344] 1. Total metaphyseal area (TV, mm²): metaphyseal area between 1.2and

[0345] 3.6 mm distal to the growth plate-epiphyseal junction.

[0346] 2. Trabecular bone area (BV, mm²): total area of trabeculaewithin TV.

[0347] 3. Trabecular bone perimeter (BS, mm): the length of totalperimeter of trabeculae.

[0348] 4. Trabecular bone volume (BV/TV, %): BV/TV×100.

[0349] 5. Trabecular bone number (TBN, #/mm): 1.199/2×BS/TV.

[0350] 6. Trabecular bone thickness (TBT, μm): (2000/1.199)×(BV/BS).

[0351] 7. Trabecular bone separation (TBS, μm): (2000×1.199)×(TV−BV).

[0352] II. Measurements and calculations related to bone resorption

[0353] 1. Osteoclast number (OCN, #): total number of osteocdast withintotal metaphyseal area.

[0354] 2. Osteoclast perimeter (OCP, mm): length of trabecular perimetercovered by osteoclast.

[0355] 3. Osteoclast number/mm (OCN/mm, #/mm): OCN/BS.

[0356] 4. Percent osteoclast perimeter (% OCP, %): OCP/BS×100.

[0357] III. Measurements and calculations related to bone formation andturnover

[0358] 1. Single-calcein labeled perimeter (SLS, mm): total length oftrabecular perimeter labeled with one calcein label.

[0359] 2. Double-calcein labeled perimeter (DLS, mm): total length oftrabecular perimeter labeled with two calcein labels.

[0360] 3. Inter-labeled width (ILW, μm): average distance between twocalcein labels.

[0361] 4. Percent mineralizing perimeter (PMS, %): (SLS/2+DLS)/BS×100.

[0362] 5. Mineral apposition rate (MAR, μm/day): ILW/label interval.

[0363] 6. Bone formation rate/surface ref. (BFR/BS, μm²/d/μm):(SLS/2+DLS)×MAR/BS.

[0364] 7. Bone turnover rate (BTR, %/y): (SLS/2+DLS)×MAR/BV×100.

[0365] Statistics

[0366] Statistics can be calculated using StatView 4.0 packages (AbacusConcepts, Inc., Berkeley, Calif.). The analysis of variance (ANOVA) testfollowed by Fishers PLSD can be used to compare the differences betweengroups.

ESTROGEN AGONIST/ANTAGONIST PROTOCOL

[0367] Estrogen agonist/antagonists are a class of compounds whichinhibits bone turnover and prevents estrogen deficiency induced boneloss. The ovariectomized rat bone loss model has been widely used as amodel of postmenopausal bone loss. Using this model, one can test theefficacy of the estrogen agonist/antagonist compounds in preventing boneloss and inhibiting bone resorption.

[0368] Sprague-Dawley female rats (Charles River, Wilmington, Mass.) atdifferent ages (such as 5 months of age) are used in these studies. Therats are singly housed in 20cm×32 cm×20 cm cages during the experimentalperiod. All rats are allowed free access to water and a pelletedcommercial diet (Agway ProLab 3000, Agway County Food, Inc., Syracuse,N.Y.) containing 0.97% calcium, 0.85% phosphorus, and 1.05 IU/g ofVdt.D₃

[0369] A group of rats (8 to 10) are sham-operated and treated p.o. withvehicle (10% ethanol and 90% saline, 1 ml/day), while the remaining ratsare bilaterally ovariectomized (OVX) and treated with either vehicle(p.o.), 17β-estradiol (Sigma, E-8876, E₂, 30 μg/kg, daily subcutaneousinjection), or estrogen agonist/antagonists (such as droloxifene at 5,10, or 20 mg/kg, daily p.o.) for a certain period (such as 4 weeks). AlIrats are given subcutaneous injections of 10 mg/kg calcein (fluorochromebone marker) 12 and 2 days before being sacrificed in order to examinethe dynamic changes in bone tissue. After 4 weeks of treatment, the ratsare autopsied. The following endpoints are determined:

[0370] Body Weight Gain

[0371] body weight at autopsy minus body weight at surgery.

[0372] Uterine Weight and Histology

[0373] The uterus is removed from each rat during autopsy, and weighedimmediately. Thereafter, the uterus is processed for histologicmeasurements such as uterine cross-sectional tissue area, stromalthickness, and luminal epithelial thickness.

[0374] Total Serum Cholesterol

[0375] Blood is obtained by cardiac puncture and allowed to clot at 4°C., and then centrifuged at 2,000 g for 10 min. Serum samples areanalyzed for total serum cholesterol using a high performancecholesterol calorimetric assay (Boehringer Mannheim Biochemicals,Indianapolis, Ind.).

[0376] Femoral Bone Mineral Measurements

[0377] The right femur from each rat is removed at autopsy and scannedusing dual energy x-ray absorptiometry (DEXA, QDR 1000/W, Hologic Inc.,Waltham, Mass.) equipped with “Regional High Resolution Scan” software(Hologic Inc., Waltham, Mass.). The scan field size is 5.08×1.902 cm,resolution is 0.0254×0.0127 cm and scan speed is 7.25 mm/second. Thefemoral scan images are analyzed and bone area, bone mineral content(BMC), and bone mineral density (BMD) of whole femora (WF), distalfemoral metaphyses (DFM), femoral shaft (FS), and proximal femora (PF)is determined

[0378] Proximal Tibial Metaphyseal Cancellous Bone HistomorphometricAnalyses

[0379] The right tibia is removed at autopsy, dissected free of muscle,and cut into three parts. The proximal tibia is fixed in 70% ethanol,dehydrated in graded concentrations of ethanol, defatted in acetone,then embedded in methyl methacrylate (Eastman Organic Chemicals,Rochester, N.Y.). Frontal sections of proximal tibial metaphyses at 4and 10 μm thickness are cut using Reicher-Jung Polycut S microtome. One4 μm and one 10 μm sections from each rat are used for cancellous bonehistomorphometry. The 4 μm sections are stained with modified Masson'sTrichrome stain while the 10 μm sections remained unstained.

[0380] A Bioquant OS/2 histomorphometry system (R&M biometrics, Inc.,Nashville, Tenn.) is used for the static and dynamic histomorphometricmeasurements of the secondary spongiosa of the proximal tibialmetaphyses between 12 and 3.6 mm distal to the growth plate-epiphysealjunction. The first 12 mm of the tibial metaphyseal region is omitted inorder to restrict measurements to the secondary spongiosa The 4 μmsections are used to determine indices related to bone volume, bonestructure, and bone resorption, while the 10 μm sections are used todetermine indices related to bone formation and bone turnover.

[0381] I. Measurements and calculations related to trabecular bonevolume and structure

[0382] 1. Total metaphyseal area (TV, mm²): metaphyseal area between 1.2and 3.6 mm distal to the growth plate epiphyseal junction.

[0383] 2. Trabecular bone area (BV, mm²): total area of trabeculaewithin TV.

[0384] 3. Trabecular bone perimeter (BS, mm): the length of totalperimeter of trabeculae.

[0385] 4. Trabecular bone volume (BV/TV, %): BV/TV×100.

[0386] 5. Trabecular bone number (TBN, #/mm): 1.199/2 ×BS/TV.

[0387] 6. Trabecular bone thickness (TBT, μm): (2000/1.199)×(BV/BS).

[0388] 7. Trabecular bone separation (TBS, μm): (2000×1.199)×(TV−BV).

[0389] II. Measurements and calculations related to bone resorption

[0390] 1. Osteoclast number (OCN, #): total number of osteoclast withintotal metaphyseal area

[0391] 2. Osteoclast perimeter (OCP, mm): length of trabecular perimetercovered by osteoclast.

[0392] 3. Osteoclast number/mm (OCN/mm, #/mm): OCN/BS.

[0393] 4. Percent osteoclast perimeter (% OCP, %): OCP/BS×100.

[0394] Ill. Measurements and calculations related to bone formation andturnover

[0395] 1. Single-calcein labeled perimeter (SLS, mm): total length oftrabecular perimeter labeled with one calcein label.

[0396] 2. Double-calcein labeled perimeter (DLS, mm): total length oftrabecular perimeter labeled with two calcein labels.

[0397] 3. Inter-labeled width (ILW, μm): average distance between twocalcein labels. 4. Percent mineralizing perimeter (PMS, %):(SLS/2+DLS)/BS×100.

[0398] 5. Mineral apposition rate (MAR, μm/day): ILW/label interval.

[0399] 6. Bone formation rate/surface ref. (BFR/BS, μm²/d/μm):(SLS/2+DLS)×MAR/BS.

[0400] 7. Bone turnover rate (BTR, %/y): (SLS/2+DLS)×MAR/BV×100.

[0401] Statistics

[0402] Statistics are calculated using StatView 4.0 packages (AbacusConcepts, Inc., Berkeley, Calif.). The analysis of variance (ANOVA) testfollowed by Fisher's PLSD is used to compare the differences betweengroups.

ANABOLIC AGENT PROTOCOL

[0403] The activity of anabolic bone agents in stimulating boneformation and increasing bone mass can be tested in intact male orfemale rats, sex hormone deficient male (orchidectomy) or female(ovariectomy) rats.

[0404] Male or female rats at different ages (such as 3 months of age)are used in the study. The rats are either intact or castrated(ovariectomized or orchidectomized), and subcutaneously injected ororally treated with anabolic agents such as prostaglandin E2 (PGE2) atdifferent doses (such as 1, 3, or 6 mg/kg/day) for certain periods (suchas 2 weeks to 2 months). In the castrated rats, treatment is started atthe next day after surgery (for the purpose of preventing bone loss) orat the time bone loss has already occurred (for the purpose of restoringbone mass). During the study, all rats are allowed free access to waterand a pelleted commercial diet (Teklad Rodent Diet #8064, Harlan Teklad,Madison, Wis.) containing 1.46% calcium, 0.99% phosphorus and 4.96 IU/gof Vit.D₃. All rats are given subcutaneous injections of 10 mg/kgcalcein on days 12 and 2 before sacrifice.

[0405] The rats are sacrificed. The following endpoints are determined:

[0406] Femoral Bone Mineral Measurements

[0407] The right femur from each rat is removed at autopsy and scannedusing dual energy x-ray absorptiometry (DEXA, QDR 1000/W, Hologic Inc.,Waltham, Mass.) equipped with “Regional High Resolution Scan” software(Hologic Inc., Waltham, Mass.). The scan field size is 5.08×1.902 cm,resolution is 0.0254×0.0127 cm and scan speed is 7.25 mm/second. Thefemoral scan images are analyzed and bone area. bone mineral content(BMC), and bone mineral density (BMD) of whole femora (WF), distalfemoral metaphyses PFM), femoral shaft (FS), and proximal femora (PF)are determined

[0408] Proximal Tibial Metaphvseal Cancellous Bone HistomorphometricAnalyses

[0409] The right tibia is removed at autopsy, dissected free of muscle,and cut into three parts. The proximal tibia is fixed in 70% ethanol,dehydrated in graded concentrations of ethanol, defatted in acetone,then embedded in methyl methacrylate (Eastman Organic Chemicals,Rochester, N.Y.). Frontal sections of proximal tibial metaphyses at 4and 10 μm thickness are cut using Relchert-Jung Polycut S microtome. One4 μm and one 10 μm sections from each rat are used for cancellous bonehistomorphometry. The 4 μm sections are stained with modified Masson'sTrichrome stain while the 10 μm sections remained unstained.

[0410] A Bioquant OS/2 histomorphometry system (R&M biometrics, Inc.,Nashville, Tenn.) is used for the static and dynamic histomorphometricmeasurements of the secondary spongiosa of the proximal tibialmetaphyses between 1.2 and 3.6 mm distal to the growth plate-epiphysealjunction. The first 1.2 mm of the tibial metaphyseal region are omittedin order to restrict measurements to the secondary spongiosa. The 4 μmsections are used to determine indices related to bone volume, bonestructure, and bone resorption, while the 10 μm sections are used todetermine indices related to bone formation and bone turnover.

[0411] I. Measurements and calculations related to trabecular bonevolume and stricture

[0412] 1. Total metaphyseal area (TV, mm²): metaphyseal area between 1.2and 3.6 mm distal to the growth plate-epiphyseal junction.

[0413] 2. Trabecular bone area (BV, mm²: total area of trabeculae withinTV.

[0414] 3. Trabecular bone perimeter (BS, mm): the length of totalperimeter of trabeculae.

[0415] 4. Trabecular bone volume (BV/TV, %): BV/TV×100.

[0416] 5. Trabecular bone number (TBN, #/mm): 1.199/2×BS/TV.

[0417] 6. Trabecular bone thickness (TBT, μm): (2000/1.199)×(BV/BS).

[0418] 7. Trabecular bone separation (TBS, μm): (2000×1.199)×(TV−BV).

[0419] II. Measurements and calculations related to bone resorption

[0420] 1. Osteoclast number (OCN, #): total number of osteoclast withintotal metaphyseal area.

[0421] 2. Osteoclast perimeter (OCP, mm): length of trabecular perimetercovered by osteociast.

[0422] 3. Osteoclast number/mm (OCN/mm, #/mm): OCN/BS.

[0423] 4. Percent osteoclast perimeter (% OCP, %): OCP/BS×100.

[0424] III. Measurements and calculations related to bone formation andturnover

[0425] 1. Single-calcein labeled perimeter (SLS, mm): total length oftrabecular perimeter labeled with one calcein label.

[0426] 2. Doublecalcein labeled perimeter (DLS, mm): total length oftrabecular perimeter labeled with two calcein labels.

[0427] 3. lnterlabeled width (ILW, μm): average distance between twocalcein labels.

[0428] 4. Percent mineralizing perimeter (PMS, %): (SLS/2+DLS)/BS×100.

[0429] 5. Mineral apposition rate (MAR, μm/day): ILW/label Interval.

[0430] 6. Bone formation rate/surface ref. (BF/RIBS, μm²/d/μm):(SLS/2+DLS)×MAR/BS.

[0431] 7. Bone turnover rate (BTR, %/y): (SLS/2+DLS)×MAR/BV×100.

[0432] Statistics

[0433] Statistics are calculated using StatView 4.0 packages (AbacusConcepts, Inc., Berkeley, Calif.). The analysis of variance (ANOVA) testfollowed by Fishers PLSD is used to compare the differences betweengroups.

IN VITRO ESTROGEN RECEPTOR BINDING ASSAY

[0434] An in vitro estrogen receptor binding assay, which measures theability of the estrogen agonist/antagonist compounds of the presentinvention to displace [3H]-estradiol from human estrogen receptorobtained by recombinant methods in yeast, is used to determine theestrogen receptor binding affinity of the compounds of this invention.The materials used in this assay are: (1) Assay buffer, TD-0.3(containing 10 Nm Tris, pH 7.6, 0.3 M potassium chloride and 5 mMDithiothreitol (DTT) (Sigma Co.), pH 7.6); (2) The radioligand used is[3H]-estradiol obtained from New England Nuclear; (3) the cold ligandused is estradiol obtained from Sigma (4) recombinant human estrogenreceptor, hER.

[0435] A solution of the compound being tested is prepared in TD-0.3with 4% DMSO and 16% ethanol. The tritiated estradiol is dissolved inTD-0.3 such that the final concentration in the assay is 5 nM. The hERis also diluted with TD-0.3 such that 4-10 μg of total protein is ineach assay well. Using microtitre plates, each incubate receives 50 ulof cold estradiol (nonspecific binding) or the compound solution, 20 uLof the tritiated estradiol and 30 ul of hER solutions. Each platecontains in triplicate total binding and varying concentrations of thecompound. The plates are incubated overnight at 4° C. The bindingreaction is then terminated by the addition and mixing of 100 mL of 3%hydroxylapatite in 10 mM tris, pH 7.6 and incubation for 15 minutes at4° C. The mixtures are centrifuged and the pellet washed four times with1% Triton-X100 in 10 mM Tris, pH 7.6. The hydroxylapatite pellets aresuspended in Ecoscint A and radioactivity is assessed using betascintigraphy. The mean of all triplicate data points (counts per minute,cpm's) is determined. Specific binding is calculated by subtractingnonspecific cpm's (defined as counts that remain following separation ofreaction mixture containing recombinant receptor, radioligand, andexcess unlabeled ligand) from total bound cpm's (defined as counts thatremain following the separation of reaction mixture containing onlyrecombinant receptor, radioligand). Compound potency is determined bymeans of IC50 determinations (the concentration of a compound needed toinhibition 50% of the of the total specific trtiated estradiol bound).Specific binding in the presence of varying concentrations of compoundis determined and calculated as percent specific binding of totalspecific radioligand bound. Data are plotted as percent inhibition bycompound (linear scale) versus compound concentration (log scale).

GROWTH HORMONE/GROWTH HORMONE SECRETAGOGUE PROTOCOL

[0436] Compounds that have the ability to stimulate GH secretion fromcultured rat pituitary cells are identified using the followingprotocol. This test is also useful for comparison to standards todetermine dosage levels. Cells are isolated from pituitaries of 6-weekold male Wistar rats. Following decapitation, the anterior pituitarylobes are removed into cold, sterile Hank's balanced salt solutionwithout calcium or magnesium (HBSS). Tissues are finely minced, thensubjected to two cycles of mechanically assisted enzymatic dispersionusing 10 U/mL bacterial protease (EC 3.4.24.4, Sigma P-6141) in HBSS.The tissue-enzyme mixture is stirred in a spinner flask at 30 rpm in a5% CO2 atmosphere at about 37° C. for about 30 min, with manualtrituration after about 15 min and about 30 min using a 10 mL pipet.This mixture is centrifuged at 200×g for about 5 min. Horse serum isadded to the supernatant to neutralize excess protease. The pellet isresuspended in fresh protease, stirred for about 30 min more under theprevious conditions, and manually triturated, ultimately through a23-gauge needle. Again, horse serum is added, then the cells from bothdigests are combined, pelleted (200×g for about 15 min), washed,resuspended in culture medium and counted. Cells are plated at6.0-6.5×104 cells per cm² in 48-well Costar dishes and cultured for 3-4days in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 4.5g/L glucose, 10% horse serum, 2.5% fetal bovine serum, 1% non-essentialamino acids, 100 U/mL nystatin and 50 mg/mL gentarnycin sulfate beforeassaying for GH secretion.

[0437] Just prior to assay, culture wells are rinsed twice, thenequilibrated for about 30 minutes in release medium (D-MEM buffered with25 mM Hepes, pH 7.4 and containing 0.5% bovine serum albumin at 37° C.).Test compounds are dissolved in DMSO, then diluted into pre-warmedrelease medium. Assays are run in quadruplicate. The assay is initiatedby adding 0.5 mL of release medium (with vehicle or test compound) toeach culture well. Incubation is carried out at about 37° C. for about15 minutes, then terminated by removal of the culture medium, which iscentrifuged at 2000×g for about 15 minutes to remove cellular material.Rat growth hormone concentrations in the supernatants are determined bya standard radioimmunoassay protocol using a rat growth hormonereference preparation (NIDDK-rGH-RP-2) and rat growth hormone antiserumraised in monkey (NlDDK-anti-rGH-S-5) obtained from Dr. A. Parlow(Harbor-UCLA Medical Center, Torrence, Calif.). Additional rat growthhormone (1.5 U/mg, #G2414, Scripps Labs, San Diego, Calif.) is iodinatedto a specific activity of approximately 30 μCi/μg by the chloramine Tmethod for use as tracer. Immune complexes are obtained by adding goatantiserum to monkey IgG (Organon Teknika, Durham, N.C.) pluspolyethylene glycol, MW 10,000-20,000 to a final concentration of 4.3%;recovery is accomplished by centrifugation. This assay has a workingrange of 0.08-2.5 μg rat growth hormone per tube above basal levels.Active compounds typically stimulate growth hormone release by greaterthan 1.4 fold. Reference: Cheng, K, Chan, W. -S., Barreto, Jr., A.,Convey, E. M., Smith, R. G. 1989.

[0438] Assay for Exogenously-Simulated Growth Hormone Release in the Ratafter Intravenous Administration of Test Compounds

[0439] Twenty-one day old female Sprague-Dawley rats (Charles RiverLaboratory, Wilmington, Mass.) are allowed to acclimate to localvivarium conditions (24° C., 12 hr light, 12 hr dark cycle) forapproximately 1 week before compound testing. All rats are allowedaccess to water and a pelleted commercial diet (Agway Country Food,Syracuse N.Y.) ad libitum.

[0440] On the day of the experiment, test compounds are dissolved invehicle containing 1% ethanol, 1 mM acetic acid and 0.1% bovine serumalbumin in saline. Each compound is tested with n=3. Rats are weighedand anesthetized via intraperitoneal injection of sodium pentobarbftal(Nembutol, 50 mg/kg body weight). Fourteen minutes after anestheticadministration, a blood sample is taken by nicking the tip of the talland allowing the blood to drip into a microcentrifuge tube (baselineblood sample, approximately 100 μl). Fifteen minutes after anestheticadministration, test compound is delivered by intravenous injection intothe tail vein, with a total injection volume of 1 ml/kg body weight.Additional blood samples are taken from the tail at 5, 10 and 15 minutesafter compound administration. Blood samples are kept on ice until serumseparation by centrifugatlon (1430×g for 10 minutes at 10° C.). Serum isstored at −80° C. until serum growth hormone determination byradio-immunoassay as described above and below.

[0441] Assessment of Exogenously-Stimulated Growth Hormone Release inthe Dog after Oral Administration

[0442] On the day of experimentation, the test compound is weighed outfor the appropriate dose and dissolved in water. Doses are delivered ata volume of 0.5 ml/kg by gavage to 4 dogs for each dosing regimen. Bloodsamples (2 ml) are collected from the jugular vein by direct venapuncture pro-dose and at 0.08, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, and 8hours post dose using 2 ml vacutainers containing lithium heparin. Theprepared plasma is stored at −20° C. until analysis.

[0443] Measurement of Canine Growth Hormone

[0444] Canine growth hormone concentrations are determined by a standardradioimmunoassay protocol using canine growth hormone (antigen foriodination and reference preparation AFP-1983B) and canine growthhormone antiserum raised in monkey (AFP-21452578) obtained from Dr. AParlow (HarborUCLA Medical Center, Torrence, Calif.). Tracer is producedby chloramine Tiodination of canine growth hormone to a specificactivity of 20-40 μCi/μg. Immune complexes are obtained by adding goatantiserum to monkey IgG (Organon Teknika, Durham, N.C.) pluspolyethylene glycol, MW 10,000-20,000 to a final concentration of 4.3%;recovery is accomplished by centrifugation. This assay has a workingrange of 0.08-2.5 μg canine GH/tube.

[0445] Administration of the compounds of this invention can be via anymethod which delivers a compound of this invention systemically and/orlocally. These methods include oral routes, parenteral, intraduodenalroutes, etc. Generally, the compounds of this invention are administeredorally, but parenteral administration (e.g., intravenous, intramuscular,subcutaneous or intramedullary) may be utilized, for example, where oraladministration is inappropriate for the instant target or where thepatient is unable to ingest the drug. The two different compounds ofthis invention can be co-administered simultaneously or sequentially inany order, or a single pharmaceutical composition comprising a firstcompound as described above and a second compound as described above ina pharmaceutically acceptable carrier can be administered.

[0446] For example, the bone anabolic agent can be used alone or incombination with an antiresorptive agent for three months to threeyears, followed by an anti-resorptive agent alone for three months tothree years, with optional repeat of the full treatment cycle.Alternatively, for example, the bone anabolic agent can be used alone orin combination with an anti-resorptive agent for three months to threeyears, followed by an anti-resorptive agent alone for the remainder ofthe patients life. For example, in one preferred mode of administrationa second compound as described above (e.g., PGE₂) may be administeredonce daily and a first compound as described above (e.g., estrogenagonist/antagonist) may be administered daily in single or multipledoses. Alternatively, for example, in another preferred mode ofadministration the two compounds may be administered sequentiallywherein the second compound as described above (e.g., PGE₂) may beadministered once daily for a period of time sufficient to augment bonemass to a level which is above the bone fracture threshold (World HealthOrganization Study “Assessment of Fracture Risk and its Application toScreening for Postmenopausal Osteoporosis (1994). Report of a WorldHealth Organization Study Group. World Health Organization TechnicalSeries 843”) followed by administration of a first compound, asdescribed above (e.g., estrogen agonist/antagonist), daily in single ormultiple doses. It is preferred that the second compound as describedabove (e.g., PGE₂) is administered once daily in a rapid delivery formsuch as oral delivery (e.g., sustained release delivery form ispreferably avoided).

[0447] In any event the amount and timing of compounds administeredwill, of course, be dependent on the subject being treated, on theseverity of the affliction, on the manner of administration and on thejudgement of the prescribing physician. Thus, because of patient topatient variability, the dosages given below are a guideline and thephysician may titrate doses of the drug to achieve the activity (e.g.,bone mass augmentation) that the physician considers appropriate for theindividual patient. In considering the degree of activity desired, thephysician must balance a variety of factors such as bone mass startinglevel, age of the patient, presence of preexisting disease, as well aspresence of other diseases (e.g., cardiovascular). For example, theadministration of an estrogen agonist/antagonist can providecardiovascular benefits particularly, for post-menopausal women. Thefollowing paragraphs provide preferred dosage ranges for the variouscomponents of this invention.

[0448] The amount of the antresorptive agent to be used is determined byits activity as a bone loss inhibiting agent. This activity isdetermined by means of an individual compound's pharmacokinetics and itsminimal maximal effective dose in inhibition of bone loss using aprotocol as described above (ESTROGEN AGONIST/ANTAGONIST PROTOCOL).

[0449] In general an effective dosage for the activities of thisinvention, for example the bone resorption activities of this invention,for the first compounds of this invention is in the range of 0.01 to 200mg/kg/day, preferably 0.5 to 100 mg/kg/day.

[0450] In particular, an effective dosage for droloxifene is in therange of 0.1 to 40 mg/kg/day, preferably 0.1 to 5 mg/kg/day.

[0451] In particular, an effective dosage for raloxifene is in the rangeof 0.1 to 100 mg/kg/day, preferably 0.1 to 10 mg/kg/day.

[0452] In particular, an effective dosage for tamoxifen is in the rangeof 0.1 to 100 mg/kg/day, preferably 0.1 to 5 mg/kg/day.

[0453] In particular, an effective dosage for

[0454]Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0455](−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0456]Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;

[0457]Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene:

[0458]1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0459]Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;or

[0460]1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline.

[0461] is in the range of 0.0001 to 100 mg/kg/day, preferably 0.001 to10 mg/kg/day.

[0462] In particular, an effective dosage for 4-hydroxy tamoxifen is inthe range of 0.0001 to 100 mg/kg/day, preferably 0.001 to 10 mg/kg/day.

[0463] In general an amount of a bone anabolic agent (e.g., PGE₂) isused that is sufficient to augment bone mass to a level which is abovethe bone fracture threshold (as detailed in the World HealthOrganization Study previously cited herein).

[0464] In general an effective dosage for the bone anabolic agentdescribed above is in the range of 0.001 to 100 mg/kg/day, preferably0.1 to 10 mg/kg/day.

[0465] In particular, an effective dosage for PGE₂ is in the range of0.001 to 10 mg/kg/day, preferably 0.01 to 1 mg/kg/day.

[0466] In particular, an effective dosage for3S-(3-Hydroxphenyl-butyl)-2R-[6-(2H-tetrazol-5-yl)-hexyl]-cyclopentanoneis in the range of 0.001 to 20 mg/kg/day, preferably 0.01 to 10mg/kg/day.

[0467] In particular, an effective dosage for sodium fluoride is in therange of 0.01 to 50 mg/kg/day, preferably 0.2 to 10 mg/kg/day.

[0468] In particular, an effective dosage for a parathyroid hormone andmetabolites and fragments thereof is in the range of 0.00001 mg/kg/dayto 1 mg/kg/day, preferably 0.001 to 0.5 mg/kg/day.

[0469] In particular, an effective dosage for growth hormone or growthhormone secretagogues is in the range of 0.0001 to 100 mg/kg/day,preferably 0.01 to 5 mg/kg/day.

[0470] The compounds of the present invention are generally administeredin the form of a pharmaceutical composition comprising at least one ofthe compounds of this invention together with a pharmaceuticallyacceptable vehicle or diluent. Thus, the compounds of this invention canbe administered individually or together in any conventional oral,parenteral or transdermal dosage form.

[0471] For oral administration a pharmaceutical composition can take theform of solutions, suspensions, tablets, pills, capsules, powders, andthe like. Tablets containing various excipients such as sodium citrate,calcium carbonate and calcium phosphate are employed along with variousdisintegrants such as starch and preferably potato or tapioca starch andcertain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type are also employed as fillers in soft and hard-filledgelatin capsules; preferred materials in this connection also includelactose or milk sugar as well as high molecular weight polyethyleneglycols. When aqueous suspensions and/or elixirs are desired for oraladministration, the compounds of this invention can be combined withvarious sweetening agents, flavoring agents, coloring agents,emulsifying agents and/or suspending agents, as well as such diluents aswater, ethanol, propylene glycol, glycerin and various like combinationsthereof.

[0472] For purposes of parenteral administration, solutions in sesame orpeanut oil or in aqueous propylene glycol can be employed, as well assterile aqueous solutions of the corresponding water-soluble salts. Suchaqueous solutions may be suitably buffered, if necessary, and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseaqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal injection purposes. Inthis connection, the sterile aqueous media employed are all readilyobtainable by standard techniques well-known to those skilled in theart.

[0473] For purposes of transdermal (e.g.,topical) administration, dilutesterile, aqueous or partially aqueous solutions (usually in about 0.1%to 5% concentration), otherwise similar to the above parenteralsolutions, are prepared.

[0474] Methods of preparing various pharmaceutical compositions with acertain amount of active ingredient are known, or will be apparent inlight of this disclosure, to those skilled in this art. For examples,see Remington's Pharmaceutical Sciences, Mack Publishing Company,Easter, Pa., 15th Edition (1975).

[0475] Pharmaceutical compositions according to the invention maycontain 0.1-95% of the compound(s) of this invention, preferably 1%-70%.In any event, the composition or formulation to be administered willcontain a quantity of a compound(s) according to the invention in anamount effective to treat the disease/condition of the subject beingtreated.

[0476] Since the present invention relates to the augmentation andmaintenance of bone mass by treatment with a combination of activeingredients which may be administered separately, the invention alsorelates to combining separate pharmaceutical compositions in kit form.The kit includes two separate pharmaceutical compositions: an estrogenagonist/antagonist and an anabolic agent. The kit includes containermeans for containing the separate compositions such as a divided bottleor a divided foil packet. Typically the kit includes directions for theadministration of the separate components. The kit form is particularlyadvantageous when the separate components are preferably administered indifferent dosage forms (e.g., oral and parenteral), are administered atdifferent dosage intervals, or when titration of the individualcomponents of the combination is desired by the prescribing physician.

[0477] An example of such a kit is a so-called blister pack. Blisterpacks are well known in the packaging industry and are being widely usedfor the packaging of pharmaceutical unit dosage forms (tablets,capsules, and the like). Blister packs generally consist of a sheet ofrelatively stiff material covered with a foil of a preferablytransparent plastic material. During the packaging process recesses areformed in the plastic foil. The recesses have the size and shape of thetablets or capsules to be packed. Next, the tablets or capsules areplaced in the recesses and the sheet of relatively stiff material issealed against the plastic foil at the face of the foil which isopposite from the direction in which the recesses were formed. As aresult, the tablets or capsules are sealed in the recesses between theplastic foil and the sheet. Preferably the strength of the sheet is suchthat the tablets or capsules can be removed from the blister pack bymanually applying pressure on the recesses whereby an opening is formedin the sheet at the place of the recess. The tablet or capsule can thenbe removed via said opening.

[0478] It is desirable to provide a memory aid on the card, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen which the tablets or capsules sospecified should be ingested. Another example of such a memory aid is acalendar printed on the card e.g., as follows “First Week, Monday,Tuesday, . . . etc . . . . Second Week, Monday, Tuesday , . . .” etc.Other variations of memory aids will be readily apparent. A “daily dose”can be a single tablet or capsule or several pills or capsules to betaken on a given day. Also a daily dose of bone anabolic agent canconsist of one tablet or capsule while a daily dose of a anti-resorptiveagent can consist of several tablets or capsules. The memory aid shouldreflect this.

[0479] In another specific embodiment of the invention a dispenserdesigned to dispense the daily doses one at a time in the order of theirintended use is provided. Preferably, the dispenser is equipped with amemory-aid, so as to further facilitate compliance with the regimen. Anexample of such a memory-aid is a mechanical counter which indicates thenumber of daily doses that has been dispensed. Another example of such amemory-aid is a battery-powered micro-chip memory coupled with a liquidcrystal readout, or audible reminder signal which, for example, readsout the date that the last daily dose has been taken and/or reminds onewhen the next dose is to be taken.

EXAMPLE

[0480] One hundred and four female Sprague-Dawley rats (Charles River,Wilmington, Mass.) at 12 months of age were sham-operated orovariectomized (OVX) at month 0. Three months post-surgery, OVX ratswere treated with either Prostaglandin E₂ (PGE₂), a known anabolic boneagent, at 3 mg/kg/day (subcutaneously injection), or PGE₂ at 3 mg/kg/day(subcutaneously injection) combined with droloxifene (DRO) at 10mg/kg/day (orally) for 2 months. Thereafter, the PGE₂ treatment waswithdrawn and the rats were then treated with either vehicle (10%alcohol in saline) or DRO (10 mg/kg/day, orally) for another one and ahalf months as described in the following.

[0481] Group I: Eight rats were autopsied at month 0 as basal controls.

[0482] Group II: Eight sham-operated rats were autopsied at month 3 aspre-treatment controls.

[0483] Group IlIl: Eight sham-operated rats were orally treated withvehicle (10% ethanol in saline) from months 3 to 5, and autopsied atmonth 5.

[0484] Group IV: Eight sham-operated rats were orally treated withvehicle (10% ethanol in saline) from months 3 to 6.5, and autopsied atmonth 6.5.

[0485] Group V: Eight OVX rats were autopsied at month 3 aspre-treatment controls.

[0486] Group VI: Eight OVX rats were orally treated with vehicle (10%ethanol in saline) from months 3 to 5, and autopsied at month 5.

[0487] Group VII: Eight OVX rats were orally treated with vehicle (10%ethanol in saline) from months 3 to 6.5, and autopsied at month 6.5.

[0488] Group VIII: Eight OVX rats were subcutaneously injected with 3mg/kg/day of PGE₂ from months 3 to 5, and autopsied at month 5.

[0489] Group IX: Eight OVX rats were subcutaneously injected with 3mg/kg/day of PGE2 from months 3 to 5, and vehicle from 5 to 6.5 months,and then autopsied at month 6.5.

[0490] Group X: Eight OVX rats were subcutaneously injected with 3mg/kg/day of PGE₂ from months 3 to 5, and 10 mg/kg/day of DRO orallyfrom 5 to 6.5 months, and then autopsied at month 6.5.

[0491] Group XI: Eight OVX rats were subcutaneously injected with 3mg/kg/day of PGE₂ and 10 mg/kg/day of DRO orally from months 3 to 5, andthen autopsied at month 5.

[0492] Group XII: Eight OVX rats were subcutaneously injected with 3mg/kg/day of PGE₂ and 10 mg/kg/day of DRO orally from months 3 to 5, andvehicle from months 5 to 6.5, then autopsied at month 6.5.

[0493] Group XIII: Eight OVX rats were subcutaneously injected with 3mg/kg/day of PGE₂ and 10 mg/kg/day of DRO orally from months 3 to 5, andDRO alone from months 5 to 6.5, then autopsied at month 6.5.

[0494] Both PGE₂ (Cayman Chemical Co., Ann Arbor, Mich.) or droloxifene(Pfizer Inc. Groton, Conn.) powder was first dissolved in 100% ethanoland further diluted with saline into desired concentrations (finalethanol concentration was 10%). A PGE₂ solution was daily injectedsubcutaneously on the back at 1 ml/kg. A droloxifene solution was givendaily p.o. at 1 ml/rat.

[0495] Lumbar Vertebral Bone Mineral Measurements

[0496] Dual energy x-ray absorptiometry (QDR 1000/W, Hologic, Inc.,Waltham, Mass.) equipped with a “Regional High Resolution Scan” software(Hologic, Inc., Waltham, Mass.) was used to determined the bone area,bone mineral content (BMC), and bone mineral density (BMD) of wholelumbar spine and each of the six lumbar vertebrae (LV1-6) in theanesthetized rats. The rats were anesthetized by injection (i.p.) of 1ml/kg of a mixture of ketamine/rompun (ratio of 4 to 3), and then placedon the rat platform. The scan field sized was 6×1.9 cm, resolution was0.0254×0.0127 cm, and scan speed was 7.25 mm/sec. The whole lumbar spinescan image was obtained and analyzed. Bone area (BA), and bone mineralcontent (BMC) were determined, and bone mineral density was calculated(MBC divided by BA) for the whole lumbar spine and each of the sixlumbar vertebrae (LV1-6).

[0497] At 3, 5, or 6.5 months post-surgery, BMC and BMD of whole lumbarspine and each of the lumbar vertebrae was significantly decreased by15% to 27% in OVX rats compared to sham controls. Rats 3 months post-OVXtreated with either PGE₂ alone or combined with DRO for 2 months hadcompletely restored BMC and BMD back to the sham control levels. Therewas no difference in BMC and BMD of OVX rats treated with PGE₂ alone orPGE₂ combined with DRO, indicating that DRO did not blunt the anaboliceffects of PGE₂. Upon PGE₂ cessation of treatment, a significantdecrease in BMD of LV1, LV2, and LV3, and in BMC of LV2 was observed. Onthe other hand, when DRO treatment was given to these OVX rats afterdiscontinuation of PGE₂, the PGE₂-restored bone was completelymaintained. Similarly, discontinuation of both PGE₂ and DRO for 1.5months produced a significant decrease in BMD of LV3. However, when PGE₂was withdrawn and DRO treatment was continued for another 1.5 months, nobone loss was found in the lumbar spine of these OVX rats. We concludedthat DRO, an anti-resorptive agent, did not blunt the anabolic effectsof PGE₂ in osteogenic rats. Further, DRO was efficacious in maintainingPGE₂-restored bone after discontinuation of PGE₂. These data support thestrategy of using an anabolic agent to restore bone mass in theosteoporotic skeleton followed by an anti-resorptive agent to maintainthe restored bone mass.

[0498] It should be understood that the invention is not limited to theparticular embodiments described herein, but that various changes andmodifications may be made without departing from the spirit and scope ofthis novel concept as defined by the following claims.

1. A pharmaceutical composition comprising: a. a therapeuticallyeffective amount of a first compound, said first compound being anestrogen agonist/antagonist; and b. a therapeutically effective amountof a second compound, said second compound being a prostaglandin or aprostaglandin agonist/antagonist.
 2. A pharmaceutical composition asrecited in claim 1 additionally comprising a pharmaceutical carrier. 3.A pharmaceutical composition as recited in claim 2 wherein the estrogenagonist/antagonist is droloxifene, raloxifene, tamoxifen,4-hydroxy-tamoxifen,Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;(−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthaiene-2-ol;Cis1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;1-(4′-Pyrrolidinoethoxyphenyl)-2-(4-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8tetrahydronaphthalene-2-ol;or1-(4′-pyrrolidinolethoxyphenyl)-2-phenylhydroxy-1,2,3,4-tetrahydroisoquinoline.4. A pharmaceutical composition according to claim 3 wherein the secondcompound is PGD₁, PGD₂, PGE₂, PGE₁, PGF₂, PGF₂α or3S-(3-Hydroxyphenyl-butyl)-2R-[6-(1H-tetrazol-5-yl)-hexyl]-cyclopentanone.5. A pharmaceutical composition as recited in claim 4 wherein theestrogen agonist/antagonist is droloxifene.
 6. A pharmaceuticalcomposition according to claim 5 wherein the second compound is PGE₂. 7.A pharmaceutical composition according to claim 5 wherein the secondcompound is3S-(3-Hydroxy-4-phenyl-butyl)-2R-[6-(2H-tetrazol-5-yl)-hexyl]-cyclopentanone.8. A pharmaceutical composition according to claim 4 wherein theestrogen agonist/antagonist isCis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;(−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene2-ol;Cis1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;1-(4′-Pyrrolidinoethoxyphonyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin1-yl-ethoxy)-phenyl]-5,6,7,8tetrahydronaphthaiene-2-ol;or1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4tetrahydroisoquinoline.9. A pharmaceutical composition according to claim 8 wherein the secondcompound is PGE₂.
 10. A pharmaceutical composition according to claim 8wherein the second compound is3S(3-Hydroxyphenyl-butyl)-2R-[6-(2H-tetrazol-5-yl)-hexyl-4-]-cyclopentanone.11. A method for treating a mammal having a condition which presentswith low bone mass comprising administering to a mammal having acondition which presents with low bone mass a. a therapeuticallyeffective amount of a first compound, said first compound being anestrogen agonist/antagonist; and b. a therapeutically effective amountof a second compound, said second compound being a prostaglandin or aprostaglandin agonist/antagonist.
 12. A method as recited in claim 11wherein the estrogen agonist/antagonist is droloxifene, raloxifene,tamoxifen, 4-hydroxy-tamoxifen, idoxifene, centrachroman,Cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;(−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthaiene-2-ol;Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetraydrohaphthalene;1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetahydronaphthalene-2-ol;or1-(4′-Pyrrolidinolethoxyphonyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroquinoline.13. A method as recited in claim 12 wherein the second compound is PGD₁,PGD₂, PGE₂, PGE₁, PGF₂, PGF₂α or3S-(3H-Hydroxy-4-phenyl-butyl)-2R-[6-(1H-tetrazol-5-yl)-hexyl]-cyclopentanone.
 14. A method as recited in claim13 wherein the estrogen agonist/antagonist is droloxifene.
 15. A methodas recited in claim 14 wherein the second compound is PGE₂.
 16. A methodas recited in claim 14 wherein the second compound is3S3-Hydroxy-4-phenyl-butyl)-2R-[6-(1H-tetrazol-5-yl)]-hexyl-cyclopentanone.17. A method as recited in claim 14 wherein the condition which presentswith low bone mass is osteoporosis.
 18. A method as recited in claim 13wherein the estrogen agonist/antagonist isCis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;(−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;or1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline.19. A method as recited in claim 18 wherein the second compound is PGE₂.20. A method as recited in claim 18 wherein the second compound is3S-(3-Hydroxy-4-phenyl-butyl)-2R-[6-(1H-tetrazol-5-yl)-hexyl]-cyclopentanone.
 21. A method as recited in claim18 wherein the condition which presents with low bone mass isosteoporosis.
 22. A method as recited in claim 14 wherein the firstcompound and the second compounds are administered substantiallysimultaneously.
 23. A method as recited in claim 14 wherein the secondcompound is administered for a period of from about three months toabout three yews.
 24. A method as recited in claim 23 followed byadministration of the first compound for a period of from about threemonths to about three years without the administration of the secondcompound during the period of from about three months to about threeyears.
 25. A method as recited in claim 23 followed by administration ofthe first compound for a period greater than about three years withouttho administration of the second compound during the greater than aboutthree year period.
 26. A method as recited in claim 18 wherein the firstcompound and the second compounds are administered substantiallysimultaneously.
 27. A method as recited in claim 18 wherein the secondcompound is administered for a period of from about three months toabout three yews.
 28. A method as recited in claim 27 followed byadministration of the first compound for a period of from about threemonths to about three years without the administration of the secondcompound during the period of from about three months to about threeyears.
 29. A method as recited in claim 27 followed by administration ofthe first compound for a period greater than about three years withoutthe administration of the second compound during the greater than aboutthree year period.
 30. A method for treating mammals which present withlow bone mass comprising administering to a mammal having a conditionwhich presents with low bone mass the pharmaceutical composition ofclaim 1 .
 31. A pharmaceutical composition comprising a. an amount of afirst compound, said first compound being an estrogenagonist/antagonist; and b. an amount of a second compound, said secondcompound being a prostaglandin or a prostaglandin agonist/antagonistwherein the amount of the first compound alone and the amount of thesecond compound alone is insufficient to achieve therapeutic effects ofincrease in bone formation and decrease in bone resorption ifadministered simultaneously and wherein the combined effect of theamount of the first and second compounds is greater than the sum of thetherapeutic effect achievable with the individual amounts of the firstand second compound, and a pharmaceutically acceptable diluent orcarrier.
 32. A method for treating a mammal having a condition whichpresents with low bone mass comprising administering to a mammal havinga condition which presents with low bone mass a. an amount of a firstcompound, said first compound being an estrogen agonist/antagonist; andb. an amount of a second compound, said second compound being aprostaglandin or a prostaglandin agonist/antagonist wherein the amountof the first compound alone and the amount of the second compound aloneis insufficient to achieve the therapeutic effects of increase in boneformation and decrease in bone resorption if administered simultaneouslyand wherein the combined effect of the amounts of the first and secondcompounds is greater than the sum of the therapeutic effects achievablewith the individual amounts of the first and second compound, and apharmaceutically acceptable diluent or carrier.
 33. A kit containing atreatment for a condition which presents with low bone mass comprising:a. a therapeutically effective amount of an estrogen agonist/antagonistand a pharmaceutically acceptable carrier in a first unit dosage form;b. a therapeutically effective amount of a prostaglandin or aprostaglandin agonist/antagonist and a pharmaceutically acceptablecarrier in a second unit dosage form; and c. container means forcontaining said first and second dosage forms.
 34. A pharmaceuticalcomposition comprising: a. a therapeutically effective amount of a firstcompound, said first compound being droloxifene, raloxifene, tamoxifenor idoxifene; and b. a therapeutically effective amount of a secondcompound, said second compound being sodium fluoride orN-[1(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677.35. A pharmaceutical composition as recited in claim 34 additionallycomprising a pharmaceutical carrier.
 36. A method for treating a mammalhaving a condition which presents with low bone mass comprisingadministering to a mammal having a condition which presents with lowbone mass a. a therapeutically effective amount of a first compound,said first compound being droloxifene, raloxifene, tamoxifen oridoxifene; and b. a therapeutically effective amount of a secondcompound, said second compound being sodium fluoride orN-[1(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677.37. A method as recited in claim 36 wherein the condition which presentswith low bone mass is osteoporosis.
 38. A method as recited in claim 36wherein the first compound and the second compounds are administeredsubstantially simultaneously.
 39. A method as recited in claim 36wherein the second compound is administered for a period of from aboutthree months to about three years.
 40. A method as recited in claim 39followed by administration of the first compound for a period of fromabout three months to about three years without the administration ofthe second compound during the period of from about three months toabout three years.
 41. A method as recited in claim 39 followed byadministration of the first compound for a period greater than aboutthree years without the administration of the second compound during thegreater than about three year period.
 42. A method for treating mammalswhich present with low bone mass comprising administering to a mammalhaving a condition which presents with low bone mass the pharmaceuticalcomposition of claim 34 .
 43. A pharmaceutical composition comprising Lan amount of a first compound, said first compound being droloxifene,raloxifene, tamoxifen or idoxifene; and b. an amount of a secondcompound, said second compound being sodium fluoride orN-[1(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-methylpropanamide:MK-677wherein the amount of the first compound alone and the amount of thesecond compound alone is insufficient to achieve the therapeutic effectsof increase in bone formation and decrease in bone resorption ifadministered simultaneously and wherein the combined effect of theamounts of the first and second compounds is greater than the sum of thetherapeutic effects achievable with the individual amounts of the firstand second compound, and a pharmaceutically acceptable diluent orcarrier.
 44. A method for treating a mammal having a condition whichpresents with low bone mass comprising administering to a mammal havinga condition which presents with low bone mass a. an amount of a firstcompound, said first compound being droloxifene, raloxifene, tamoxifenor idoxifene; and b. an amount of a second compound, said secondcompound being sodium fluoride orN-[1-(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2amino-2-methylpropanamide:MK-M667wherein the amount of the first compound alone and the amount of thesecond compound alone is insufficient to achieve the therapeutic effectsof increase in bone formation and decrease in bone resorption ifadministered simultaneously and wherein the combined effect of theamounts of the first and second compounds is greater than the sum of thetherapeutic effects achievable with the individual amounts of the firstand second compound, and a pharmaceutically acceptable diluent orcarrier.
 45. A kit containing a treatment for a condition which presentswith low bone mass comprising: a. a therapeutically effective amount ofdroloxifene, raloxifene, tamoxifen or idoxifene and a pharmaceuticallyacceptable carrier in a first unit dosage form; b. a therapeuticallyeffective amount of a sodium fluoride orN-[1(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677and a pharmaceutically acceptable carrier in a second unit dosage form;and c. container means for containing said first and second dosageform:.
 46. A pharmaceutical composition comprising: a. a therapeuticallyeffective amount of a first compound, said first compound beingCis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2ol;(−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinolne;Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;or1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;and b. a therapeutically effective amount of a second compound, saidsecond compound being sodium fluoride, a parathyroid hormone, growthhormone or a growth hormone secretagogue.
 47. A pharmaceuticalcomposition as recited in claim 46 additionally comprising apharmaceutical carrier.
 48. A pharmaceutical composition as recited inclaim 47 wherein the second compound is sodium fluoride.
 49. Apharmaceutical a composition as recited in claim 47 wherein the secondcompound is a parathyroid hormone.
 50. A pharmaceutical composition asrecited in claim 47 wherein the second compound is growth hormone.
 51. Apharmaceutical composition as recited in claim 47 wherein the secondcompound is a growth hormone secretagogue.
 52. A method for treating amammal having a condition which presents with low bone mass comprisingadministering to a mammal having a condition which presents with lowbone mass a. a therapeutically effective amount of a first compound,said first compound beingCis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthaiene-2-ol;(−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene2-olor1-(4′-Pyrrolidinolethoxyphenyl)-2-phenylthydroxy-1,2,3,4-tetrahydroisoquinoline;and b. a therapeutically effective amount of a second compound, saidsecond compound being sodium fluoride, a parathyroid hormone, growthhormone or a growth hormone secretagogue.
 53. A method as recited inclaim 52 wherein the second compound is sodium fluoride.
 54. A method asrecited in claim 52 wherein the second compound is a parathyroidhormone.
 55. A method as recited in claim 52 wherein the second compoundis growth hormone.
 56. A method as recited in claim 52 wherein thesecond compound is a growth hormone secretagogue.
 57. A method asrecited in claim 52 wherein the condition which presents with low bonemass is osteoporosis.
 58. A method as recited in claim 52 wherein thefirst compound and the second compound are administered substantiallysimultaneously.
 59. A method as recited in claim 52 wherein the secondcompound is administered for a period of from about three months toabout three years.
 60. A method as recited in claim 59 followed byadministration of the first compound for a period of from about threemonths to about three years without the administration of the secondcompound during the period of from about three months to about threeyears.
 61. A method as recited in claim 59 followed by administration ofthe first compound for a period greater than about three years withoutthe administration of the second compound during the greater than aboutthree year period.
 62. A method for treating mammals which present withlow bone mass comprising administering to a mammal having a conditionwhich presents with low bone mass the pharmaceutical composition ofclaim 46 .
 63. A pharmaceutical composition comprising a. an amount of afirst compound, said first compound beingCis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;(−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;1-(4′-Pyrroldinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;or1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;b. an amount of a second compound, said second compound being sodiumfluoride, a parathyroid hormone, growth hormone or a growth hormonesecretagogues wherein the amount of the first compound alone and theamount of the second compound alone is insufficient to achieve thetherapeutic effects of increase in bone formation and decrease in boneresorption if administered simultaneously and wherein the combinedeffect of the amounts of the first and second compounds is greater thanthe sum of the therapeutic effects achievable with the individualamounts of the first and second compound, and a pharmaceuticallyacceptable diluent or carrier.
 64. A method for treating a mammal havinga condition which presents with low bone mass comprising administeringto a mammal having a condition which presents with low bone mass a. anamount of a first compound, said first compound beingCis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;(−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2;Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl-2-phenyl]-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;or1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroquinoline;and b. an amount of a second compound, said second compound being sodiumfluoride, a parathyroid hormone, growth hormone or a growth hormonesecretagogue wherein the amount of the first compound alone and theamount of the second compound alone is insufficient to achieve thetherapeutic effects of increase in bone formation and decrease in boneresorption if administered simultaneously and wherein the combinedeffect of the amounts of the first and second compounds is greater thanthe sum of the therapeutic effects achievable with the individualamounts of the first and second compound, and a pharmaceuticallyacceptable diluent or carrier.
 65. A kit containing a treatment for acondition which presents with low bone mass comprising: a. atherapeutically effective amount ofCis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;(−)-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene2ol;Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol;Cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydrohaphthalene;1-(4′-Pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;Cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin1-yl-ethoxy)phenyl]-5,6,7,8-tetrahydronaphthaiene2-ol; or1-(4′-Pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinolineand a pharmaceutically acceptable carrier in a first unit dosage form;b. a therapeutically effective amount of sodium fluoride, a parathyroidhormone, growth hormone or a growth hormone secretagogue and apharmaceutically acceptable carrier in a second unit dosage form; and c.container means for containing said first and second dosage forms. 66.The pharmaceutical composition as recited in claim 34 wherein the firstcompound is droloxifene.
 67. The method as recited in claim 36 whereinthe compound is droloxifene.
 68. The method as recited in claim 40wherein the first compound is droloxifene.
 69. The method as recited inclaim 41 wherein the first compound is droloxifene.
 70. Thepharmaceutical composition as recited in claim 43 wherein the firstcompound is droloxifene.
 71. The method as recited in claim 44 whereinthe first compound is droloxifene.
 72. The kit as recited in claim 45wherein the first compound is droloxifene.
 73. A pharmaceuticalcomposition comprising: a. a therapeutically effective amount of a firstcompound, said first compound being raloxifene, tamoxifen or idoxifene;and b. a therapeutically effective amount of a second compound, saidsecond compound being a parathyroid hormone, growth hormone or a growthhormone secretagogue.
 74. A pharmaceutical composition as recited inclaim 73 additionally comprising a pharmaceutical carrier.
 75. Apharmaceutical composition as recited in claim 74 wherein the firstcompound is raloxifene.
 76. A pharmaceutical a composition as recited inclaim 74 wherein the second compound is a parathyroid hormone.
 77. Apharmaceutical composition as recited in claim 74 wherein the secondcompound is growth hormone.
 78. A pharmaceutical composition as recitedin claim 74 wherein the second compound is a growth hormonesecretagogue.
 79. A method for treating a mammal having a conditionwhich presents with low bone mass comprising administering to a mammalhaving a condition which presents with low bone mass a. atherapeutically effective amount of a first compound, said firstcompound being raloxifene, tamoxifen or idoxifene; and b. atherapeutically effective amount of a second compound, said secondcompound being a parathyroid hormone, growth hormone or a growth hormonesecretagogue.
 80. A method as recited in claim 79 wherein the firstcompound is raloxifene.
 81. A method as recited in claim 79 wherein thesecond compound is a parathyroid hormone.
 82. A method as recited inclaim 79 wherein the second compound is growth hormone.
 83. A method asrecited in claim 79 wherein the second compound is a growth hormonesecretagogue.
 84. A method as recited in claim 79 wherein the conditionwhich presents with low bone mass is osteoporosis.
 85. A method asrecited in claim 79 wherein the first compound and the second compoundare administered substantially simultaneously.
 86. A method as recitedin claim 79 wherein the second compound is administered for a period offrom about three months to about three years.
 87. A method as recited inclaim 86 followed by administration of the first compound for a periodof from about three months to about three years without theadministration of the second compound during the period of from aboutthree months to about three years.
 88. A method as recited in claim 86followed by administration of the first compound for a period greaterthan about three years without the administration of the second compoundduring the greater than about three year period.
 89. A method fortreating mammals which present with low bone mass comprisingadministering to a mammal having a condition which presents with lowbone mass the pharmaceutical composition of claim 73 .
 90. Apharmaceutical composition comprising a. an amount of a first compound,said first compound being raloxifene, tamoxifen or idoxifene; or b. anamount of a second compound, said second compound being a parathyroidhormone, growth hormone or a growth hormone secretagogues wherein theamount of the first compound alone and the amount of the second compoundalone is insufficient to achieve the therapeutic effects of increase inbone formation and decrease in bone resorption if administeredsimultaneously and wherein the combined effect of the amounts of thefirst and second compounds is greater than the sum of the therapeuticeffects achievable with the individual amounts of the first and secondcompound, and a pharmaceutically acceptable diluent or carrier.
 91. Amethod for treating a mammal having a condition which presents with lowbone mass comprising administering to a mammal having a condition whichpresents with low bone mass a. an amount of a first compound, said firstcompound being raloxifene, tamoxifen or idoxifene; and b. an amount of asecond compound, said second compound being a parathyroid hormone,growth hormone or a growth hormone secretagogue wherein the amount ofthe first compound alone and the amount of the second compound alone isinsufficient to achieve the therapeutic effects of increase in boneformation and decrease in bone resorption if administered simultaneouslyand wherein the combined effect of the amounts of the first and secondcompounds is greater than the sum of the therapeutic effects achievablewith the individual amounts of the first and second compound, and apharmaceutically acceptable diluent or carrier.
 92. A kit containing atreatment for a condition which presents with low bone mass comprising:a. a therapeutically effective amount of raloxifene, tamoxifen oridoxifene; and a pharmaceutically acceptable carrier in a first unitdosage form; b. a therapeutically effective amount of a parathyroldhormone, growth hormone or a growth hormone secretagogue and apharmaceutically acceptable carrier in a second unit dosage form; and c.container means for containing said first and second dosage forms.